Methods of using molecular conjugates comprising monoclonal antibodies to dendritic cells

ABSTRACT

The present invention provides novel methods for targeting an antigen to a dendritic cell using an antibody linked to the antigen. The present invention further provides methods of inducing or enhancing an immune response and methods of immunization using an antibody linked to the antigen.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/035,637 filed Nov. 7, 2001, which is a continuation-in-partapplication of U.S. application Ser. No. 09/851,614 filed May 8, 2001,which claims priority to U.S. Provisional Application No. 60/230,739filed on Sep. 7, 2000, and U.S. Provisional Application No. 60/203,126,filed on May 8, 2000, the entire contents of which are herebyincorporated by reference herein.

BACKGROUND OF THE INVENTION

Dendritic cells are specialized cells of the immune system with theunique capacity for initiating primary and secondary T and B lymphocyteresponses. Characterized as professional antigen presenting cells(APCs), dendritic cells express MHC and costimulatory moleculesessential in priming naïve T lymphocytes. This unique property ofdendritic cells, also termed “nature's adjuvant”, has led to a greatinterest in their possible role of autoimmune diseases as well as theirpotential for exploitation in immunotherapy of various diseases.

Recent advances in culturing of dendritic cells has greatly increasedour understanding of this complex type of cells. There are several typeof dendritic cells that are distinguished by their lineage, location intissues, phenotype, and function. The dendritic cells type that mostprominently associates with T lymphocytes for the initiation of immuneresponses is of bone marrow origin. Bone marrow-derived dendritic cellscan be further segregated into 1) thymic dendritic cells, which are oflymphoid origin and appear to be involved specifically in deletion ofmaturing T lymphocytes, 2) Langerhans cells, which are of myeloidlineage and have specialized APC function in the skin, and 3) myeloidlineage-derived dendritic cells found particularly in the blood, spleenand lymph nodes.

The hallmarks of myeloid lineage-derived dendritic cells (includingLangerhans cells) are the following: 1) capacity for antigen uptake, andprocessing for presentation, 2) capacity for selective migration intissues, and 3) capacity for direct stimulation of T lymphocytes (bothnaïve and primed).

Despite the recent advances in characterization of dendritic cells, verylittle is known regarding dendritic cell specific receptors ormolecules. There are numerous dendritic cell-associated molecules thatare shared with other myeloid and non-myeloid cells, however, verylimited reagents are available which are specific to dendritic cells.Reagents, in particular antibodies, which react specifically orpreferentially with dendritic cells have great potential as targetingagents to induce potent immune responses to tumor or infectious diseaseantigens. These cell-specific targeting agents could also be engineeredto deliver toxins to eliminate potent APCs (e.g., dendritic cells) inbone marrow and organ transplantations or other autoimmune disorders.

Accordingly, dendritic cell-specific binding agents would be of greattherapeutic and diagnostic value.

SUMMARY OF THE INVENTION

The present invention provides isolated human monoclonal antibodieswhich bind to antigen presenting cells (APCs), particularly humandendritic cells, as well as vaccine conjugates, bispecific molecules,immunotoxins and therapeutic compositions containing such antibodies.Accordingly, the antibodies and compositions of the invention can beused in a variety of dendritic cell-targeted therapies, for example toenhance antigen presentation or to treat APC-mediated diseases.

In certain embodiments, the human antibodies are characterized by highaffinity binding to dendritic cells, and by their ability to affectdendritic cell growth and/or function by targeting molecules or cellswith defined functions (e.g., a tumor cell, a bacterium, a virus, aneffector cell) to dendritic cells. Accordingly, the human monoclonalantibodies of the invention can be used as diagnostic or therapeuticagents in vivo and in vitro.

In other embodiments, the human antibodies are characterized by bindingto particular novel epitopes (e.g., receptors) on dendritic cells. Forexample, specific antibodies of the present invention include humanmonoclonal antibody B11 comprising the heavy and light chain amino acidsequences shown in SEQ ID NOS: 2 and 4, respectively, which binds to thehuman macrophage mannose receptor (also referred to herein as “human B11antigen”) having an approximate molecular weight of 180 kD as measuredby SDS-PAGE and comprising the amino acid sequence shown in SEQ ID NO:7.Another specific antibody of the present invention is human monoclonalantibody E21 which binds to the human dendritic cell E21 antigen havingan approximate molecular weight of 36-40 kD as measured by SDS-PAGE.

Isolated human antibodies of the invention encompass various antibodyisotypes, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD,and IgE. Typically, they include IgG1 (e.g., IgG1κ) and IgM isotypes.The antibodies can be full-length (e.g., an IgG1 or IgG4 antibody) orcan include only an antigen-binding portion (e.g., a Fab, F(ab′)₂, Fv ora single chain Fv fragment). In one embodiment, the human antibodies arerecombinant human antibodies. In another embodiment, the humanantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic non-human animal, e.g., a transgenic mouse, having agenome comprising a human heavy chain transgene and a human light chaintransgene fused to an immortalized cell. Particular human antibodies ofthe invention include those produced by hybridomas referred to herein asA3, A5, A23, A24, A33, B9, B11, B33, B47, C8, C10, C20, C28, C29, C30,C35, E1, E8, E10, E18, E20, E21, E24, 10F7, 28-6, 24-3, 25-14, 17-6,19-2 and 19-4.

In another embodiment, human antibodies of the present invention arecharacterized by specific binding to human dendritic cells and one ormore of the following properties:

-   -   a) the ability to bind to the mannose receptor present on human        dendritic cells with a binding equilibrium association constant        (Ka) of at least about 10⁷ M⁻¹;    -   b) the ability to opsonize human dendritic cells;    -   c) the ability to be internalized after binding to human        dendritic cells; and    -   d) the ability to block binding to the mannose receptor on human        dendritic cells.

Accordingly, isolated human antibodies of the invention typically bindto dendritic cells with a binding equilibrium association constant (Ka)of at least about 10⁷ M⁻¹, preferably about 10⁸ M⁻¹, more preferably,about 10⁹ M⁻¹, and more preferably about 10¹⁰ to 10¹¹ M⁻¹ or higher.

In another aspect, the invention provides nucleic acid moleculesencoding the antibodies, or antigen-binding portions, of the invention.Accordingly, recombinant expression vectors which include theantibody-encoding nucleic acids of the invention, and host cellstransfected with such vectors, are also encompassed by the invention, asare methods of making the antibodies of the invention by culturing thesehost cells.

In yet another aspect, the invention provides isolated B-cells from atransgenic non-human animal, e.g., a transgenic mouse, which are capableof expressing various isotypes (e.g., IgG, IgA and/or IgM) of humanmonoclonal antibodies that specifically bind to dendritic cells.Preferably, the isolated B cells are obtained from a transgenicnon-human animal, e.g., a transgenic mouse, which has been immunizedwith a purified or enriched preparation of dendritic cells. Preferably,the transgenic non-human animal, e.g., a transgenic mouse, has a genomecomprising a human heavy chain transgene and a human light chaintransgene. The isolated B-cells are then immortalized to provide asource (e.g., a hybridoma) of human monoclonal antibodies to dendriticcells.

Accordingly, the present invention also provides a hybridoma capable ofproducing human monoclonal antibodies that specifically bind todendritic cells. In one embodiment, the hybridoma includes a B cellobtained from a transgenic non-human animal, e.g., a transgenic mouse,having a genome comprising a human heavy chain transgene and a humanlight chain transgene, fused to an immortalized cell. The transgenicnon-human animal can be immunized with a purified or enrichedpreparation of dendritic cells to generate antibody-producinghybridomas. Particular hybridomas of the invention include thosedesignated herein as A3, A5, A23, A24, A33, B9, B11, B33, B47, C8, C10,C20, C28, C29, C30, C35, E1, E8, E10, E18, E20, E21 E24, 10F7, 28-6,24-3, 25-14, 17-6, 19-2 and 19-4.

In yet another aspect, the invention provides a transgenic non-humananimal, such as a transgenic mouse (also referred to herein as a“HuMab”), which express human monoclonal antibodies that specificallybind to dendritic cells. In a particular embodiment, the transgenicnon-human animal is a transgenic mouse having a genome comprising ahuman heavy chain transgene and a human light chain transgene. Thetransgenic non-human animal can be immunized with a purified or enrichedpreparation of dendritic cells. Preferably, the transgenic non-humananimal, e.g., the transgenic mouse, is capable of producing multipleisotypes of human monoclonal antibodies to dendritic cells (e.g., IgG,IgA and/or IgM) by undergoing V-D-J recombination and isotype switching.Isotype switching may occur by, e.g., classical or non-classical isotypeswitching.

In another aspect, the present invention provides methods for producinghuman monoclonal antibodies which specifically react with dendriticcells. In one embodiment, the method includes immunizing a transgenicnon-human animal, e.g., a transgenic mouse, having a genome comprising ahuman heavy chain transgene and a human light chain transgene, with apurified or enriched preparation of dendritic cells. B cells (e.g.,splenic B cells) of the animal are then obtained and fused with myelomacells to form immortal, hybridoma cells that secrete human monoclonalantibodies against dendritic cells.

Isolated anti-dendritic cell human monoclonal antibodies of theinvention, or antigen binding portions thereof, can be derivatized orlinked to another functional molecule, e.g., another peptide or protein(e.g., another antibody, antibody fragment, a tumor ligand or anantigen). For example, an antibody or antigen-binding portion of theinvention can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreantigens, such as a tumor antigen, autoantigen or pathogenic antigen, toform a vaccine conjugate for enhancing dendritic cell-mediated immuneresponses. Human anti-dendritic cell antibodies of the invention alsocan be linked to other therapeutic agents, such as cytotoxic drugs,enzymatically active toxins, radioisotopes, and small molecules, such asimmunomodulatory (e.g., anti-inflammatory) compounds and anti-cancerdrugs.

In a particular embodiment, the present invention provides a humananti-dendritic cell monoclonal antibody, or a fragment thereof,conjugated to a melanoma-specific antigen or a prostate cancer-specificantigen. Specific conjugates encompassed by the invention include, forexample, human anti-dendritic cell antibody B11 linked or geneticallyfused to Pmel17, as encoded by the nucleic acid sequence shown in SEQ IDNO:8. Other tumor antigens can similarly be linked or fused to B11,including for example, Gp100, PSA and PSMA.

In yet another aspect, the present invention provides a method forinducing or enhancing an immune response against an antigen (e.g., atumor cell antigen, a microbial antigen, or a viral antigen) in asubject, comprising administering to the subject a molecular complexcomprising at least one binding specificity for a component on thesurface of a dendritic cell linked to at least one antigen, wherein thecomponent on the surface of the dendritic cell mediates internalizationof the molecular complex when bound by the binding specificity. In oneembodiment, the immune response comprises antibodies that bind to theantigen. In another embodiment, the immune response comprises T cellsthat bind to the antigen as a component of an MHC-I or MHC-II complex.

In one aspect, the anti-dendritic cell antibody, or a fragment thereof,can be used to target whole cells (e.g., a tumor cell, an effector cell)or pathogens to dendritic cells for the induction of an immune response.In another aspect, a cell can be transfected or transduced with anucleic acid molecule encoding a human anti-dendritic cell antibody ofthe invention such that the anti-dendritic cell antibody is expressed onthe surface of the cell. In another aspect, a human anti-dendritic cellantibody of the invention can be directly chemically or otherwisecrosslinked, anchored or tagged to the cell surface of a cell (e.g., atumor cell, a bacterium or a virus) such that the cell can be targetedto dendritic cells.

In a further aspect, the invention provides a method for immunizing asubject, comprising administering to the subject an effective amount ofa vaccine conjugate containing a human anti-dendritic cell antibody asdescribed above.

In another aspect, the present invention features a bispecific ormultispecific molecule comprising at least one anti-dendritic cellantibody of the invention (or fragment thereof) and a second bindingspecificity for an antigen on a target cell or a pathogen. A target cellis a cell whose elimination would be beneficial to the host, e.g., atumor cell, a microbial pathogen, or a virus or virus-infected cell.Suitable target antigens include, for example, tumor-associatedantigens, pathogenic antigens, autoantigens and Fc receptors (e.g., FcγRor FcαR).

Multispecific molecules of the invention also include trispecific,tetraspecific and other multispecific molecules. In one embodiment themultispecific molecule includes an anti-enhancement factor (EF) portion,e.g., a molecule which binds to a surface protein involved in cytotoxicactivity.

In another aspect, the present invention provides compositions, e.g.,pharmaceutical and diagnostic compositions, comprising apharmaceutically acceptable carrier and at least one human monoclonalantibody of the invention, or an antigen-binding portion thereof, whichbinds to dendritic cells. In one embodiment, the composition comprises acombination of the human antibodies or antigen-binding portions thereof,preferably each of which binds to a distinct epitope. Thus, thecombination provides multiple therapies tailored to provide the maximumtherapeutic benefit. Compositions, e.g., pharmaceutical compositions,comprising a combination of at least one human monoclonal antibody ofthe invention, or a bispecific molecule, multispecific molecule, vaccineconjugate or immunotoxin including at least one human monoclonalantibody of the invention, along with one or more other therapeuticagents (e.g., cytotoxic agents or cytokines) are also within the scopeof the invention.

In yet another aspect, the invention provides a method for inhibitingthe proliferation and/or differentiation of dendritic cells byinhibiting growth and/or by inducing phagocytosis and/or killing ofdendritic cells by human effector cells, such as human polymorphonuclearcells (PMNs), monocytes and macrophages, using an antibody, orantigen-binding portion thereof (or a bispecific or multispecificantibody) of the invention. In one embodiment, the method comprisescontacting a dendritic cell either in vitro or in vivo with one or acombination of human monoclonal antibodies of the invention, or anantigen-binding portion thereof, in the presence of a human effectorcell. The method can be employed in culture, e.g. in vitro or ex vivo(e.g., cultures comprising dendritic cells and effector cells). Forexample, a sample containing dendritic cells and effector cells can becultured in vitro, and combined with an antibody of the invention, or anantigen-binding portion thereof (or a bispecific or multispecificmolecule of the invention). Alternatively, the method can be performedin a subject, e.g., as part of an in vivo (e.g., therapeutic orprophylactic) protocol.

For in vivo methods, the antibody, or antigen-binding portion thereof(or a bispecific or multispecific molecule of the invention), can beadministered to a human subject suffering from a dendritic cell-mediateddisease. These diseases include, for example, autoimmune disease,inflammatory disease, and graft versus host disease. Exemplaryautoimmune diseases that can be treated (e.g., ameliorated) or preventedusing the methods and compositions of the invention include, but are notlimited to rheumatoid arthritis, multiple sclerosis, diabetes mellitus,myasthenia gravis, pernicious anemia, Addison's disease, lupuserythematosus, Reiter's syndrome, and Graves disease.

In one embodiment, the subject can be additionally treated with an agentthat modulates, e.g., enhances or inhibits, the expression or activityof Fc receptor, e.g., an Fcγ receptor or an Fcα receptor, by forexample, treating the subject with a cytokine. Preferred cytokines foradministration during treatment with the bispecific and multispecificmolecule include granulocyte colony-stimulating factor (G-CSF),granulocyte- macrophage colony-stimulating factor (GM-CSF), interferon-γ(IFN-γ), and tumor necrosis factor (TNF).

Isolated human monoclonal antibody compositions of the invention alsocan be administered in combination with other known therapies, e.g., ananti-inflammatory or immunosuppressant therapies, or cytotoxins.

In yet another aspect, the present invention provides a method fordetecting in vitro or in vivo the presence of dendritic cells in asample, e.g., for diagnosing a dendritic cell-related disease. In oneembodiment, this is achieved by contacting a sample to be tested, alongwith a control sample, with a human monoclonal antibody of theinvention, or an antigen-binding portion thereof (or a bispecific ormultispecific molecule), under conditions that allow for formation of acomplex between the antibody and a dendritic cell. Complex formation isthen detected (e.g., using an ELISA) in both samples, and anystatistically significant difference in the formation of complexesbetween the samples is indicative the presence of dendritic cells in thetest sample. Other features and advantages of the instant invention beapparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the reactivity of human monoclonal antibodies B11, C20 andE21 with dendritic cells and hematopoietic cell lines U937, CEM, THP-1and L540, as assessed by flow cytometry. Binding was measured by meanfluorescence intensity.

FIG. 2 shows the dose-dependent binding of human monoclonal antibodiesB11, C20 and E21 to dendritic cells, as assessed by flow cytometry.Binding was measured by mean fluorescence intensity.

FIG. 3 shows the dose-dependent reactivity of human monoclonal antibodyB11 with CD34+ stem cell-derived dendritic cells, as assessed by flowcytometry. Binding was measured by mean fluorescence intensity.

FIG. 4 shows the binding of human monoclonal antibody B11 to dendriticcells and macrophages, as assessed by flow cytometry.

FIG. 5 shows the binding of human monoclonal antibody B11 to THP-1induced to differentiate into a dendritic cell phenotype, as assessed byflow cytometry.

FIG. 6 shows binding of human monoclonal antibody B11 to macaquedendritic cells, as assessed by flow cytometry. Binding was measured bymean fluorescence intensity.

FIG. 7 shows the percent internalization of human monoclonal antibodyB11 by dendritic cells over time at 37° C.

FIG. 8 shows increased antigen presentation via antibody B11 compared toantigen alone, as measured by stimulation of tetanus toxoid-specific Tcells.

FIG. 9 shows the B11 ScFv construct which was created by linking theV_(L) (SEQ ID NO:1 and 2) and V_(H) (SEQ ID NO: 3 and 4) domains ofhuman monoclonal antibody B11.

FIG. 10 shows a binding comparison between whole human monoclonalantibody B11 and F(ab′)2 fragments of B11 to dendritic cells, asmeasured by FACS analysis.

FIG. 11 shows the percent of FITC-dextran internalization by dendriticcells.

FIG. 12 shows that conjugation of antigen to B11 enhances antigenpresentation, as 10 to 100-fold lower amounts of antibody B11-conjugatedtetanus toxoid are required to achieve the same level of T cellstimulation as with tetanus toxoid alone.

FIG. 13 shows the nucleotide and corresponding amino acid sequences ofthe variable light (VL) and variable heavy (VL) chains of antibody B11.

FIG. 14 shows the vector map of vaccine conjugate MMV4 which includesmonoclonal antibody B11 fused to Pmel17 antigen (SEQ ID NO:8 and 9).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel antibody-based therapies formodulating an immune response against an antigen, and for treating anddiagnosing diseases, including diseases mediated by dendritic cells.

Therapies of the invention employ isolated human monoclonal antibodies,or antigen-binding portions thereof, which bind to an epitope present onantigen presenting cells (APC), particularly dendritic cells and cellsrelated thereto. While the human antibodies can be produced using avariety of known technique, the present invention exemplifies for thefirst time their generation in a non-human transgenic animal, e.g., atransgenic mouse, capable of producing multiple isotypes of humanmonoclonal antibodies to dendritic cells (e.g., IgG, IgA and/or IgE) byundergoing V-D-J recombination and isotype switching. Accordingly,various aspects of the invention include human antibodies, antibodyfragments, antibody mimetics, bispecific and multispecific molecules,vaccine conjugates, immunotoxins and pharmaceutical compositionsthereof, as well as non-human transgenic animals, and B-cells andhybridomas for making such monoclonal antibodies. Methods of using theantibodies of the invention to detect a dendritic cells or a relatedcell type expressing a dendritic cell antigen, or to inhibit growth,differentiation and/or activity of a dendritic cell, either in vitro orin vivo, are also encompassed by the invention.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “dendritic cell” as used herein, includes immature and maturedendritic cells and related myeloid progenitor cells that are capable ofdifferentiating into dendritic cells, or related antigen presentingcells (e.g., monocytes and macrophages) in that they express antigens incommon with dendritic cells. As used herein, the term “related” includesa cell that is derived from a common progenitor cell or cell lineage. Ina preferred embodiment, binding of an antibody of the invention to adendritic cell inhibits the growth of dendritic cells. In anotherpreferred embodiment, binding of an antibody of the invention todendritic cells mediates an effect on dendritic cell growth and/orfunction by targeting molecules or cells with defined functions (e.g.,tumor cells, effector cells, microbial pathogens) to dendritic cells. Ina further embodiment, binding of an antibody of the invention to adendritic cell results in internalization of the antibody by thedendritic cell.

As used herein, the term “antibody” refers to a glycoprotein comprisingat least two heavy (H) chains and two light (L) chains inter-connectedby disulfide bonds. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as HCVR or VH) and a heavy chainconstant region. The heavy chain constant region is comprised of threedomains, CH1, CH2 and CH3. Each light chain is comprised of a lightchain variable region (abbreviated herein as LCVR or VL) and a lightchain constant region. The light chain constant region is comprised ofone domain, CL. The VH and VL regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each VH and VL is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (Clq) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., an antigen on a dendritic cell). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consistsof a VH domain; and (vi) an isolated complementarity determining region(CDR). Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those with skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

The term “bispecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has two differentbinding specificities which bind to, or interact with, for example, (a)a dendritic cell and (b) an Fc receptor on the surface of an effectorcell. In another embodiment, a bispecific molecule of the invention hastwo different binding specificities which bind to, or interact with (a)a dendritic cell and (b) an antigen on a target cell (e.g., a tumorcell). The term “multispecific molecule” or “heterospecific molecule” isintended to include any agent, e.g., a protein, peptide, or protein orpeptide complex, which has more than two different binding specificitieswhich bind to, or interact with, for example, (a) a dendritic cell, (b)an Fc receptor on the surface of an effector cell, and (c) at least oneother component. Accordingly, the invention includes, but is not limitedto, bispecific, trispecific, tetraspecific, and other multispecificmolecules which are directed to cell surface antigens, such as adendritic cell antigen, and to Fc receptors on effector cells, or anantigen on a target cell (e.g., a tumor cell). The term “bispecificantibodies” further includes diabodies. Diabodies are bivalent,bispecific antibodies in which the VH and VL domains are expressed on asingle polypeptide chain, but using a linker that is too short to allowfor pairing between the two domains on the same chain, thereby forcingthe domains to pair with complementary domains of another chain andcreating two antigen binding sites (see e.g., Holliger, P., et al.(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al.(1994) Structure 2:1121-1123).

As used herein, the term “heteroantibodies” refers to two or moreantibodies, antibody binding fragments (e.g., Fab), derivativestherefrom, or antigen binding regions linked together, at least two ofwhich have different specificities. These different specificitiesinclude a binding specificity for a dendritic cell, and a bindingspecificity for an Fc receptor on an effector cell, or an antigen orepitope on a target cell, e.g., a tumor cell.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.Accordingly, the term “human monoclonal antibody” refers to antibodiesdisplaying a single binding specificity which have variable and constantregions derived from human germline immunoglobulin sequences. In oneembodiment, the human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic non-human animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene, fused to an immortalized cell.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from ananimal (e.g., a mouse) that is transgenic for human immunoglobulin genes(described further in Section I, below); antibodies expressed using arecombinant expression vector transfected into a host cell, antibodiesisolated from a recombinant, combinatorial human antibody library, orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences.In certain embodiments, however, such recombinant human antibodies aresubjected to in vitro mutagenesis (or, when an animal transgenic forhuman Ig sequences is used, in vivo somatic mutagenesis) and thus theamino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangermline VH and VL sequences, may not naturally exist within the humanantibody germline repertoire in vivo.

As used herein, a “heterologous antibody” is defined in relation to thetransgenic non-human organism producing such an antibody. This termrefers to an antibody having an amino acid sequence or an encodingnucleic acid sequence corresponding to that found in an organism notconsisting of the transgenic non-human animal, and generally from aspecies other than that of the transgenic non-human animal.

As used herein, a “heterohybrid antibody” refers to an antibody having alight and heavy chains of different organismal origins. For example, anantibody having a human heavy chain associated with a murine light chainis a heterohybrid antibody. Examples of heterohybrid antibodies includechimeric and humanized antibodies, discussed supra.

An “isolated antibody”, as used herein, is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to dendritic cells and related myeloid derivedantigen presenting cells (e.g., monocytes and macrophages), and issubstantially free of antibodies that specifically bind cell types otherthan dendritic cells). An isolated antibody that specifically binds to adendritic cell may, however, have cross-reactivity to other cells, e.g.,cell types that express an antigen that is related to the cognateantigen on a dendritic cell. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals. In oneembodiment of the invention, a combination of “isolated” monoclonalantibodies having different specificities are combined in a well definedcomposition.

As used herein, the terms “specific binding” and “specifically binds to”refers to specificity of antibody binding to a predetermined antigen orcell type. An antibody which “binds specifically to”, or is “specificfor” a particular antigen or cell type, binds to the antigen or celltype with selectivity over other antigens and cell types. While eachantibody of the invention binds specifically to a particular targetepitope (e.g., present on the surface of a dendritic cell), specificantibodies of the invention, in certain embodiments, may exhibit somecross-reactivity with other APCs. In other embodiments, the specificantibodies react only with dendritic cells. Typically, the antibodybinds with an affinity of at least about 1×10⁷ M⁻¹, and binds to thepredetermined antigen with an affinity that is at least two-fold greaterthan its affinity for binding to a non-specific antigen (e.g., BSA,casein) other than the predetermined antigen or a closely-relatedantigen. The phrases “an antibody recognizing an antigen” and “anantibody specific for an antigen” are used interchangeably herein withthe term “an antibody which binds specifically to an antigen”.

As used herein, the term “high affinity” for an IgG antibody refers to abinding affinity of at least about 10⁷M⁻¹, preferably at least about10⁹M⁻¹, more preferably at least about 10¹⁰M⁻¹, 10¹¹M⁻¹, 10¹²M⁻¹ orgreater, e.g., up to 10¹³M⁻¹ or greater. However, “high affinity”binding can vary for other antibody isotypes. For example, “highaffinity” binding for an IgM isotype refers to a binding affinity of atleast about 1×10⁷M⁻¹.

The term “K_(assoc)”, as used herein, is intended to refer to theassociation constant of a particular antibody-antigen interaction.

The term “K_(dis)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

As used herein, “isotype switching” refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

As used herein, “nonswitched isotype” refers to the isotypic class ofheavy chain that is produced when no isotype switching has taken place;the CH gene encoding the nonswitched isotype is typically the first CHgene immediately downstream from the functionally rearranged VDJ gene.Isotype switching has been classified as classical or non-classicalisotype switching. Classical isotype switching occurs by recombinationevents which involve at least one switch sequence region in thetransgene. Non-classical isotype switching may occur by, for example,homologous recombination between human σ_(μ) and human Σ_(μ)(δ-associated deletion). Alternative non-classical switching mechanisms,such as intertransgene and/or interchromosomal recombination, amongothers, may occur and effectuate isotype switching.

As used herein, the term “switch sequence” refers to those DNA sequencesresponsible for switch recombination. A “switch donor” sequence,typically a □ switch region, will be 5′ (i.e., upstream) of theconstruct region to be deleted during the switch recombination. The“switch acceptor” region will be between the construct region to bedeleted and the replacement constant region (e.g., γ, ε, etc.). As thereis no specific site where recombination always occurs, the final genesequence will typically not be predictable from the construct.

As used herein, “glycosylation pattern” is defined as the pattern ofcarbohydrate units that are covalently attached to a protein, morespecifically to an immunoglobulin protein. A glycosylation pattern of aheterologous antibody can be characterized as being substantiallysimilar to glycosylation patterns which occur naturally on antibodiesproduced by the species of the nonhuman transgenic animal, when one ofordinary skill in the art would recognize the glycosylation pattern ofthe heterologous antibody as being more similar to said pattern ofglycosylation in the species of the nonhuman transgenic animal than tothe species from which the CH genes of the transgene were derived.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete VH or VL domain, respectively. Arearranged immunoglobulin gene locus can be identified by comparison togermline DNA; a rearranged locus will have at least one recombinedheptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule”, as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., VH, VL,CDR3) that bind to dendritic cells, is intended to refer to a nucleicacid molecule in which the nucleotide sequences encoding the antibody orantibody portion are free of other nucleotide sequences encodingantibodies or antibody portions that bind cells other than dendriticcells, which other sequences may naturally flank the nucleic acid inhuman genomic DNA.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsodetermined using the algorithm of E. Meyers and W. Miller (Comput. Appl.Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures may be mutated, thereof inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

The term “operatively linked” or “operably linked” is intended to meanthat molecules are functionally coupled to each other in that the changeof activity or state of one molecule is affected by the activity orstate of the other molecule. A nucleic acid is “operably linked” when itis placed into a functional relationship with another nucleic acidsequence. For instance, a promoter or enhancer is operably linked to acoding sequence if it affects the transcription of the sequence. Withrespect to transcription regulatory sequences, operably linked meansthat the DNA sequences being linked are contiguous and, where necessaryto join two protein coding regions, contiguous and in reading frame. Forswitch sequences, operably linked indicates that the sequences arecapable of effecting switch recombination. Typically, two polypeptidesthat are operably linked are covalently attached through peptide bonds.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. PRODUCTION OF HUMAN ANTIBODIES TO DENDRITIC CELLS

While particularly preferred methods of generating human monoclonalantibodies (mAbs) of the invention are described in detail herein, avariety of other techniques, including conventional monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein, Nature 256: 495 (1975) also can be used. Althoughsomatic cell hybridization procedures are preferred, other techniquesfor producing monoclonal antibody can be employed e.g., viral oroncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in murine systems is a well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are well known in the art. Fusionpartners (e.g., murine myeloma cells) and fusion procedures are alsowell known.

In a preferred embodiment, human monoclonal antibodies directed againstdendritic cells are generated using transgenic mice carrying parts ofthe human immune system rather than the mouse system. These transgenicmice, referred to herein as “HuMAb” mice, contain a human immunoglobulingene miniloci that encodes unrearranged human heavy (μ and γ) and κlight chain immunoglobulin sequences, together with targeted mutationsthat inactivate the endogenous μ and κ chain loci (Lonberg, N. et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N.(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F.and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536-546). The preparationof HuMab mice is described in detail Section II below and in Taylor, L.et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al.(1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc.Natl. Acad. Sci USA 90:3720-3724; Choi et al. (1993) Nature Genetics4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al.(1994) J. Immunol. 152:2912-2920; Lonberg et al., (1994) Nature368(6474): 856-859; Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49-101; Taylor, L. et al. (1994) InternationalImmunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N. (1995) Ann. N.Y.Acad. Sci 764:536-546; Fishwild, D. et al. (1996) Nature Biotechnology14: 845-851, the contents of all of which are hereby incorporated byreference in their entirety. See further, U.S. Pat. Nos. 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016;5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay, andGenPharm International; U.S. Pat. No. 5,545,807 to Surani et al.;International Publication Nos. WO 98/24884, published on Jun. 11, 1998;WO 94/25585, published Nov. 10, 1994; WO 93/1227, published Jun. 24,1993; WO 92/22645, published Dec. 23, 1992; WO 92/03918, published Mar.19, 1992, the disclosures of all of which are hereby incorporated byreference in their entity.

HuMab Immunizations

To generate fully human monoclonal antibodies to dendritic cells, HuMabmice can be immunized with a purified or enriched preparation ofdendritic cells, as described by Lonberg, N. et al. (1994) Nature368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851 and WO 98/24884. Preferably, the mice will be 6-16 weeks of ageupon the first immunization. For example, a purified or enrichedpreparation of dendritic cells (1-10 million cells) can be used toimmunize the HuMab mice intraperitoneally. In the event thatimmunizations using a purified or enriched preparation of dendriticcells do not result in antibodies, mice can also be immunized with adendritic cell lysate to promote immune responses.

Cumulative experience with various antigens has shown that the HuMAbtransgenic mice respond best when initially immunized intraperitoneally(IP) with antigen in complete Freund's adjuvant, followed by every otherweek IP immunizations (up to a total of 6) with antigen in incompleteFreund's adjuvant. The immune response can be monitored over the courseof the immunization protocol with plasma samples being obtained byretroorbital bleeds. The plasma can be screened, for example by ELISA orflow cytometry (as described below), and mice with sufficient titers ofanti-dendritic cell human immunoglobulin can be used for fusions. Micecan be boosted intravenously with antigen 3 days before sacrifice andremoval of the spleen. It is expected that 2-3 fusions for each antigenmay need to be performed. Several mice will be immunized for eachantigen. For example, a total of twelve HuMAb mice of the HC07 and HC012strains can be immunized.

Generation of Hybridomas Producing Human Monoclonal Antibodies toDendritic Cells

The mouse splenocytes can be isolated and fused with PEG to a mousemyeloma cell line based upon standard protocols. The resultinghybridomas are then screened for the production of antigen-specificantibodies. For example, single cell suspensions of splenic lymphocytesfrom immunized mice are fused to one-sixth the number of P3X63-Ag8.653nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cellsare plated at approximately 2×10⁵ in flat bottom microtiter plate,followed by a two week incubation in selective medium containing 20%fetal Clone Serum, 18% “653” conditioned media, 5% origen (IGEN), 4 mML-glutamine, 1 mM L˜glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50mg/ml gentamycin and 1× HAT (Sigma; the HAT is added 24 hours after thefusion). After two weeks, cells are cultured in medium in which the HATis replaced with HT. Individual wells are then screened by ELISA forhuman anti-dendritic cell monoclonal IgM and IgG antibodies. Onceextensive hybridoma growth occurs, medium is observed usually after10-14 days. The antibody secreting hybridomas are replated, screenedagain, and if still positive for human IgG, anti-dendritic cellmonoclonal antibodies, can be subcloned at least twice by limitingdilution. The stable subclones are then cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

Characterization of Binding of Human Monoclonal Antibodies to DendriticCells

To characterize binding of human monoclonal dendritic cell antibodies ofthe invention, hybridomas can be screened, for example, for positivereactivy with dendritic cells by flow cytometry.

Briefly, dendritic cells are harvested and washed, then added to 96 wellplates and incubated with dilutions of hybridoma supernatants (ormonoclonal antibodies in PBS containing 0.1% Tween 80 and 20% mouseserum) at 4° C. for 1 hour. The plates are then washed, and furtherincubated with secondary antibodies (e.g. FITC or PE-labeled anti-humanIgG) for 1 hour at 4° C. After washing the cells are fixed with 1%paraformaldehyde, and analyzed. The samples can be analyzed by FACScaninstrument using light and side scatter properties to gate on singlecells. An alternative assay using fluorescence microscopy may be used(in addition to or instead of) the flow cytometry assay. Cells can bestained exactly as described above and examined by fluorescencemicroscopy. This method allows visualization of individual cells, butmay have diminished sensitivity depending on the density of the antigen.

Hybridomas that bind with high avidity to dendritic cells will besubcloned and further characterized. One clone from each hybridoma,which retains the reactivity of the parent cells (by flow cytometry),can be chosen for making a 5-10 vial cell bank stored at −140° C., andfor antibody purification.

To purify human anti-dendritic cell antibodies, selected hybridomas canbe grown in two-liter spinner-flasks for monoclonal antibodypurification. Supernatants can be filtered and concentrated beforeaffinity chromatography with protein A-sepharose (Pharmacia, Piscataway,N.J.). Eluted IgG can be checked by gel electrophoresis and highperformance liquid chromatography to ensure purity. The buffer solutioncan be exchanged into PBS, and the concentration can be determined byOD₂₈₀ using 1.43 extinction coefficient. The monoclonal antibodies canbe aliquoted and stored at −80° C.

To determine if the selected human anti-dendritic cell monoclonalantibodies bind to unique epitopes, each antibody can be biotinylatedusing commercially available reagents (Pierce, Rockford, Ill.).Competition studies using unlabeled monoclonal antibodies andbiotinylated monoclonal antibodies can be performed using flow cytometryas described above. Biotinylated monoclonal antibody binding can bedetected with a strep-avidin-alkaline phosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed. Wells of microtiter plates can be coated with 10 μg/ml ofanti-human Ig overnight at 4° C. After blocking with 5% BSA, the platesare reacted with 10 μg/ml of monoclonal antibodies or purified isotypecontrols, at ambient temperature for two hours. The wells can then bereacted with either human IgG1 or human IgM-specific alkalinephosphatase-conjugated probes. After washing, the plates are developedwith pNPP substrate (1 mg/ml), and analyzed at OD of 405-650.

Anti-dendritic cell human IgGs can be further tested for reactivity withdendritic cells by western blotting. Briefly, cell extracts fromdendritic cells can be prepared and subjected to sodium dodecyl sulfate(SDS) polyacrylamide gel electrophoresis. After electrophoresis, theseparated antigens will be transferred to nitrocellulose membranes,blocked with 20% mouse serum, and probed with the monoclonal antibodiesto be tested. Human IgG binding can be detected using anti-human IgGalkaline phosphatase and developed with BCIP/NBT substrate tablets(Sigma Chem. Co., St. Louis, Mo.).

Phagocytic and Cell Killing Activities of Human Monoclonal Antibodies toDendritic Cells

In addition to binding specifically to dendritic cells, human monoclonalanti-dendritic cell antibodies can be tested for their ability tomediate phagocytosis and killing of dendritic cells. The testing ofmonoclonal antibody activity in vitro can provide an initial screeningprior to testing in vivo models. Briefly, polymorphonuclear cells (PMN),or other effector cells, from healthy donors can be purified by FicollHypaque density centrifugation, followed by lysis of contaminatingerythrocytes. Washed PMNs, can be suspended in RPMI supplemented with10% heat-inactivated fetal calf serum and mixed with ⁵¹Cr labeleddendritic cells, at various ratios of effector cells to dendriticcells(effector cells: dendritic cells). Purified human anti-dendriticcell IgGs can then be added at various concentrations. Irrelevant humanIgG can be used as negative control. Assays can be carried out for 0-120minutes at 37° C. Samples can be assayed for cytolysis by measuring ⁵¹Crrelease into the culture supernatant. Anti-dendritic cell monoclonal canalso be tested in combinations with each other to determine whethercytolysis is enhanced with multiple monoclonal antibodies.

Human monoclonal antibodies which bind to dendritic cells also can betested in an in vivo model (e.g., in mice) to determine their efficacyin mediating phagocytosis and killing of dendritic cells. Theseantibodies can be selected, for example, based on the followingcriteria, which are not intended to be exclusive:

1.) binding to live dendritic cells;

2.) high affinity of binding to dendritic cells;

3.) binding to a unique epitope on dendritic cells (to eliminate thepossibility that monoclonal antibodies with complimentary activitieswhen used in combination would compete for binding to the same epitope);

4.) opsonization of dendritic cells;

5.) mediation of growth inhibition, phagocytosis and/or killing ofdendritic cells in the presence of human effector cells;

6.) internalization after binding to dendritic cells;

7.) binding to dendritic cells in situ (e.g., in human tissues);

8.) activation of dendritic cells (e.g., induce cytokine release,expression of immunomodulatory surface molecules (e.g., CD80 (B7.1),CD86 (B7.2), CD40, and CD54 (ICAM));

9.) binding to the human mannose receptor on dendritic cells; and

10.) binding to a dendritic cell antigen which is conserved amongprimates.

Preferred human monoclonal antibodies of the invention meet one or more,and preferably all, of these criteria. In a particular embodiment, thehuman monoclonal antibodies are used in combination, e.g., as apharmaceutical composition comprising two or more anti-dendritic cellmonoclonal antibodies or fragments thereof. For example, humananti-dendritic cell monoclonal antibodies having different, butcomplementary activities can be combined in a single therapy to achievea desired therapeutic or diagnostic effect. An illustration of thiswould be a composition containing an anti-dendritic cell humanmonoclonal antibody that is rapidly internalized by dendritic cells,combined with another human anti-dendritic cell monoclonal antibody thatinduces antigen presenting cell activities of dendritic cells, e.g.,release of immunostimulatory cytokines.

II. PRODUCTION OF TRANSGENIC NONHUMAN ANIMALS WHICH GENERATE HUMANMONOCLONAL ANTI-DENDRITIC CELL ANTIBODIES

In yet another aspect, the invention provides transgenic non-humananimals, e.g., a transgenic mice, which are capable of expressing humanmonoclonal antibodies that specifically bind to dendritic cells,preferably with high affinity. In a preferred embodiment, the transgenicnon-human animals, e.g., the transgenic mice (HuMab mice), have a genomecomprising a human heavy chain transgene and a light chain transgene. Inone embodiment, the transgenic non-human animals, e.g., the transgenicmice, have been immunized with a purified or enriched preparation ofdendritic cells and/or a dendritic cell lysate. Preferably, thetransgenic non-human animals, e.g., the transgenic mice, are capable ofproducing multiple isotypes of human monoclonal antibodies to dendriticcells (e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination andisotype switching. Isotype switching may occur by, e.g., classical ornon-classical isotype switching.

The design of a transgenic non-human animal that responds to foreignantigen stimulation with a heterologous antibody repertoire, requiresthat the heterologous immunoglobulin transgenes contain within thetransgenic animal function correctly throughout the pathway of B-celldevelopment. In a preferred embodiment, correct function of aheterologous heavy chain transgene includes isotype switching.Accordingly, the transgenes of the invention are constructed so as toproduce isotype switching and one or more of the following: (1) highlevel and cell-type specific expression, (2) functional generearrangement, (3) activation of and response to allelic exclusion, (4)expression of a sufficient primary repertoire, (5) signal transduction,(6) somatic hypermutation, and (7) domination of the transgene antibodylocus during the immune response.

Not all of the foregoing criteria need be met. For example, in thoseembodiments wherein the endogenous immunoglobulin loci of the transgenicanimal are functionally disrupted, the transgene need not activateallelic exclusion. Further, in those embodiments wherein the transgenecomprises a functionally rearranged heavy and/or light chainimmunoglobulin gene, the second criteria of functional generearrangement is unnecessary, at least for that transgene which isalready rearranged. For background on molecular immunology, see,Fundamental Immunology, 2nd edition (1989), Paul William E., ed. RavenPress, N.Y., which is incorporated herein by reference.

In certain embodiments, the transgenic non-human animals used togenerate the human monoclonal antibodies of the invention containrearranged, unrearranged or a combination of rearranged and unrearrangedheterologous immunoglobulin heavy and light chain transgenes in thegermline of the transgenic animal. Each of the heavy chain transgenescomprises at least one C_(H) gene. In addition, the heavy chaintransgene may contain functional isotype switch sequences, which arecapable of supporting isotype switching of a heterologous transgeneencoding multiple C_(H) genes in the B-cells of the transgenic animal.Such switch sequences may be those which occur naturally in the germlineimmunoglobulin locus from the species that serves as the source of thetransgene C_(H) genes, or such switch sequences may be derived fromthose which occur in the species that is to receive the transgeneconstruct (the transgenic animal). For example, a human transgeneconstruct that is used to produce a transgenic mouse may produce ahigher frequency of isotype switching events if it incorporates switchsequences similar to those that occur naturally in the mouse heavy chainlocus, as presumably the mouse switch sequences are optimized tofunction with the mouse switch recombinase enzyme system, whereas thehuman switch sequences are not. Switch sequences may be isolated andcloned by conventional cloning methods, or may be synthesized de novofrom overlapping synthetic oligonucleotides designed on the basis ofpublished sequence information relating to immunoglobulin switch regionsequences (Mills et al., Nucl. Acids Res. 15:7305-7316 (1991); Sideraset al., Intl. Immunol. 1:631-642 (1989), which are incorporated hereinby reference). For each of the foregoing transgenic animals,functionally rearranged heterologous heavy and light chainimmunoglobulin transgenes are found in a significant fraction of theB-cells of the transgenic animal (at least 10 percent).

The transgenes used to generate the transgenic animals of the inventioninclude a heavy chain transgene comprising DNA encoding at least onevariable gene segment, one diversity gene segment, one joining genesegment and at least one constant region gene segment. Theimmunoglobulin light chain transgene comprises DNA encoding at least onevariable gene segment, one joining gene segment and at least oneconstant region gene segment. The gene segments encoding the light andheavy chain gene segments are heterologous to the transgenic non-humananimal in that they are derived from, or correspond to, DNA encodingimmunoglobulin heavy and light chain gene segments from a species notconsisting of the transgenic non-human animal. In one aspect of theinvention, the transgene is constructed such that the individual genesegments are unrearranged, i.e., not rearranged so as to encode afunctional immunoglobulin light or heavy chain. Such unrearrangedtransgenes support recombination of the V, D, and J gene segments(functional rearrangement) and preferably support incorporation of allor a portion of a D region gene segment in the resultant rearrangedimmunoglobulin heavy chain within the transgenic non-human animal whenexposed to dendritic cells.

In an alternate embodiment, the transgenes comprise an unrearranged“mini-locus”. Such transgenes typically comprise a substantial portionof the C, D, and J segments as well as a subset of the V gene segments.In such transgene constructs, the various regulatory sequences, e.g.promoters, enhancers, class switch regions, splice-donor andsplice-acceptor sequences for RNA processing, recombination signals andthe like, comprise corresponding sequences derived from the heterologousDNA. Such regulatory sequences may be incorporated into the transgenefrom the same or a related species of the non-human animal used in theinvention. For example, human immunoglobulin gene segments may becombined in a transgene with a rodent immunoglobulin enhancer sequencefor use in a transgenic mouse. Alternatively, synthetic regulatorysequences may be incorporated into the transgene, wherein such syntheticregulatory sequences are not homologous to a functional DNA sequencethat is known to occur naturally in the genomes of mammals. Syntheticregulatory sequences are designed according to consensus rules, such as,for example, those specifying the permissible sequences of asplice-acceptor site or a promoter/enhancer motif. For example, aminilocus comprises a portion of the genomic immunoglobulin locus havingat least one internal (i.e., not at a terminus of the portion) deletionof a non-essential DNA portion (e.g., intervening sequence; intron orportion thereof) as compared to the naturally-occurring germline Iglocus.

In a preferred embodiment of the invention, the transgenic animal usedto generate human antibodies to dendritic cells contains at least one,typically 2-10, and sometimes 25-50 or more copies of the transgenedescribed in Example 12 of WO 98/24884 (e.g., pHC1 or pHC2) bred with ananimal containing a single copy of a light chain transgene described inExamples 5, 6, 8, or 14 of WO 98/24884, and the offspring bred with theJ_(H) deleted animal described in Example 10 of WO 98/24884, thecontents of which are hereby expressly incorporated by reference.Animals are bred to homozygosity for each of these three traits. Suchanimals have the following genotype: a single copy (per haploid set ofchromosomes) of a human heavy chain unrearranged mini-locus (describedin Example 12 of WO 98/24884), a single copy (per haploid set ofchromosomes) of a rearranged human K light chain construct (described inExample 14 of WO 98/24884), and a deletion at each endogenous mouseheavy chain locus that removes all of the functional J_(H) segments(described in Example 10 of WO 98/24884). Such animals are bred withmice that are homozygous for the deletion of the J_(H) segments(Examples 10 of WO 98/24884) to produce offspring that are homozygousfor the J_(H) deletion and hemizygous for the human heavy and lightchain constructs. The resultant animals are injected with antigens andused for production of human monoclonal antibodies against theseantigens.

B cells isolated from such an animal are monospecific with regard to thehuman heavy and light chains because they contain only a single copy ofeach gene. Furthermore, they will be monospecific with regards to humanor mouse heavy chains because both endogenous mouse heavy chain genecopies are nonfunctional by virtue of the deletion spanning the J_(H)region introduced as described in Example 9 and 12 of WO 98/24884.Furthermore, a substantial fraction of the B cells will be monospecificwith regards to the human or mouse light chains because expression ofthe single copy of the rearranged human κ light chain gene willallelically and isotypically exclude the rearrangement of the endogenousmouse κ and lambda chain genes in a significant fraction of B-cells.

The transgenic mouse of the preferred embodiment will exhibitimmunoglobulin production with a significant repertoire, ideallysubstantially similar to that of a native mouse. Thus, for example, inembodiments where the endogenous Ig genes have been inactivated, thetotal immunoglobulin levels will range from about 0.1 to 10 mg/ml ofserum, preferably 0.5 to 5 mg/ml, ideally at least about 1.0 mg/ml. Whena transgene capable of effecting a switch to IgG from IgM has beenintroduced into the transgenic mouse, the adult mouse ratio of serum IgGto IgM is preferably about 10:1. The IgG to IgM ratio will be much lowerin the immature mouse. In general, greater than about 10%, preferably 40to 80% of the spleen and lymph node B cells express exclusively humanIgG protein.

The repertoire will ideally approximate that shown in a non-transgenicmouse, usually at least about 10% as high, preferably 25 to 50% or more.Generally, at least about a thousand different immunoglobulins (ideallyIgG), preferably 10⁴ to 10⁶ or more, will be produced, dependingprimarily on the number of different V, J and D regions introduced intothe mouse genome. These immunoglobulins will typically recognize aboutone-half or more of highly antigenic proteins, e.g., dendritic cellproteins. Typically, the immunoglobulins will exhibit an affinity forpreselected antigens of at least about 10⁷M⁻¹, preferably at least about10⁹M⁻¹, more preferably at least about 1010M⁻¹, 10¹¹M⁻¹, 10¹²M⁻¹, orgreater, e.g., up to 10¹³M⁻¹ or greater.

In some embodiments, it may be preferable to generate mice withpredetermined repertoires to limit the selection of V genes representedin the antibody response to a predetermined antigen type. A heavy chaintransgene having a predetermined repertoire may comprise, for example,human VH genes which are preferentially used in antibody responses tothe predetermined antigen type in humans. Alternatively, some VH genesmay be excluded from a defined repertoire for various reasons (e.g.,have a low likelihood of encoding high affinity V regions for thepredetermined antigen; have a low propensity to undergo somatic mutationand affinity sharpening; or are immunogenic to certain humans). Thus,prior to rearrangement of a transgene containing various heavy or lightchain gene segments, such gene segments may be readily identified, e.g.by hybridization or DNA sequencing, as being from a species of organismother than the transgenic animal.

The transgenic mice of the present invention can be immunized with apurified or enriched preparation of dendritic cells and/or a dendriticcells lysate as described previously. The mice will produce B cellswhich undergo class-switching via intratransgene switch recombination(cis-switching) and express immunoglobulins reactive with dendriticcells. The immunoglobulins can be human sequence antibodies, wherein theheavy and light chain polypeptides are encoded by human transgenesequences, which may include sequences derived by somatic mutation and Vregion recombinatorial joints, as well as germline-encoded sequences;these human sequence immunoglobulins can be referred to as beingsubstantially identical to a polypeptide sequence encoded by a humanV_(L) or V_(H) gene segment and a human J_(L) or J_(L) segment, eventhough other non-germline sequences may be present as a result ofsomatic mutation and differential V-J and V-D-J recombination joints.With respect to such human sequence antibodies, the variable regions ofeach chain are typically at least 80 percent encoded by human germlineV, J, and, in the case of heavy chains, D, gene segments; frequently atleast 85 percent of the variable regions are encoded by human germlinesequences present on the transgene; often 90 or 95 percent or more ofthe variable region sequences are encoded by human germline sequencespresent on the transgene. However, since non-germline sequences areintroduced by somatic mutation and VJ and VDJ joining, the humansequence antibodies will frequently have some variable region sequences(and less frequently constant region sequences) which are not encoded byhuman V, D, or J gene segments as found in the human transgene(s) in thegermline of the mice. Typically, such non-germline sequences (orindividual nucleotide positions) will cluster in or near CDRs, or inregions where somatic mutations are known to cluster.

The human sequence antibodies which bind to the predetermined antigencan result from isotype switching, such that human antibodies comprisinga human sequence γ chain (such as γ1, γ2a, γ2B, or γ3) and a humansequence light chain (such as K) are produced. Such isotype-switchedhuman sequence antibodies often contain one or more somatic mutation(s),typically in the variable region and often in or within about 10residues of a CDR) as a result of affinity maturation and selection of Bcells by antigen, particularly subsequent to secondary (or subsequent)antigen challenge. These high affinity human sequence antibodies mayhave binding affinities of at least 1×10⁹ M⁻¹, typically at least 5×10⁹M⁻¹, frequently more than 1×10¹⁰ M⁻¹, and sometimes 5×10¹⁰ M⁻¹ to 1×10¹¹M⁻¹ or greater.

Another aspect of the invention pertains to the B cells from such micewhich can be used to generate hybridomas expressing human monoclonalantibodies which bind with high affinity (e.g., greater than 2×10⁹ M⁻¹)to dendritic cells. Thus, in another embodiment of the invention, thesehybridomas are used to generate a composition comprising animmunoglobulin having an affinity constant (Ka) of at least 2×10⁹ M⁻¹for binding dendritic cells, wherein said immunoglobulin comprises:

a human sequence light chain composed of (1) a light chain variableregion having a polypeptide sequence which is substantially identical toa polypeptide sequence encoded by a human V_(L) gene segment and a humanJ_(L) segment, and (2) a light chain constant region having apolypeptide sequence which is substantially identical to a polypeptidesequence encoded by a human C_(L) gene segment; and

a human sequence heavy chain composed of a (1) a heavy chain variableregion having a polypeptide sequence which is substantially identical toa polypeptide sequence encoded by a human V_(H) gene segment, optionallya D region, and a human J_(H) segment, and (2) a constant region havinga polypeptide sequence which is substantially identical to a polypeptidesequence encoded by a human C_(H) gene segment.

The development of high affinity human monoclonal antibodies againstdendritic cells is facilitated by a method for expanding the repertoireof human variable region gene segments in a transgenic mouse having agenome comprising an integrated human immunoglobulin transgene, saidmethod comprising introducing into the genome a V gene transgenecomprising V region gene segments which are not present in saidintegrated human immunoglobulin transgene. Often, the V region transgeneis a yeast artificial chromosome comprising a portion of a human V_(H)or V_(L) (V_(κ)) gene segment array, as may naturally occur in a humangenome or as may be spliced together separately by recombinant methods,which may include out-of-order or omitted V gene segments. Often atleast five or more functional V gene segments are contained on the YAC.In this variation, it is possible to make a transgenic mouse produced bythe V repertoire expansion method, wherein the mouse expresses animmunoglobulin chain comprising a variable region sequence encoded by aV region gene segment present on the V region transgene and a C regionencoded on the human Ig transgene. By means of the V repertoireexpansion method, transgenic mice having at least 5 distinct V genes canbe generated; as can mice containing at least about 24 V genes or more.Some V gene segments may be non-functional (e.g., pseudogenes and thelike); these segments may be retained or may be selectively deleted byrecombinant methods available to the skilled artisan, if desired.

Once the mouse germline has been engineered to contain a functional YAChaving an expanded V segment repertoire, substantially not present inthe human Ig transgene containing the J and C gene segments, the traitcan be propagated and bred into other genetic backgrounds, includingbackgrounds where the functional YAC having an expanded V segmentrepertoire is bred into a mouse germline having a different human Igtransgene. Multiple functional YACs having an expanded V segmentrepertoire may be bred into a germline to work with a human Ig transgene(or multiple human Ig transgenes). Although referred to herein as YACtransgenes, such transgenes when integrated into the genome maysubstantially lack yeast sequences, such as sequences required forautonomous replication in yeast; such sequences may optionally beremoved by genetic engineering (e.g., restriction digestion andpulsed-field gel electrophoresis or other suitable method) afterreplication in yeast in no longer necessary (i.e., prior to introductioninto a mouse ES cell or mouse prozygote). Methods of propagating thetrait of human sequence immunoglobulin expression, include breeding atransgenic mouse having the human Ig transgene(s), and optionally alsohaving a functional YAC having an expanded V segment repertoire. BothV_(H) and V_(L) gene segments may be present on the YAC. The transgenicmouse may be bred into any background desired by the practitioner,including backgrounds harboring other human transgenes, including humanIg transgenes and/or transgenes encoding other human lymphocyteproteins. The invention also provides a high affinity human sequenceimmunoglobulin produced by a transgenic mouse having an expanded Vregion repertoire YAC transgene. Although the foregoing describes apreferred embodiment of the transgenic animal of the invention, otherembodiments are contemplated which have been classified in fourcategories:

I. Transgenic animals containing an unrearranged heavy and rearrangedlight immunoglobulin transgene;

II. Transgenic animals containing an unrearranged heavy and unrearrangedlight immunoglobulin transgene;

III. Transgenic animal containing rearranged heavy and an unrearrangedlight immunoglobulin transgene; and

IV. Transgenic animals containing rearranged heavy and rearranged lightimmunoglobulin transgenes.

Of these categories of transgenic animal, the preferred order ofpreference is as follows II>I>III>IV where the endogenous light chaingenes (or at least the K gene) have been knocked out by homologousrecombination (or other method) and I>II>III>IV where the endogenouslight chain genes have not been knocked out and must be dominated byallelic exclusion.

III. BISPECIFIC/MULTISPECIFIC MOLECULES WHICH BIND TO DENDRITIC CELLS

In yet another embodiment of the invention, human monoclonal antibodiesto dendritic cells, or antigen-binding portions thereof, can bederivatized or linked to another functional molecule, e.g., anotherpeptide, protein or other binding agent (e.g., an antibody, antibodyfragment or other ligand) to generate a bispecific or multispecificmolecule which binds both to dendritic cells and to one or more otherbinding sites or target epitopes. For example, the human antibodies ofthe invention or fragments thereof can be functionally linked (e.g., bychemical coupling, genetic fusion, noncovalent association or otherwise)to one or more other antibodies, antibody fragments, peptides (e.g.,ligands, such as a tumor ligand) or binding mimetics which bind to apathogen or cell other than a dendritic cell, such as a tumor cell, sothat the pathogen or cell is targeted to dendritic cells.

Accordingly, the present invention includes bispecific and multispecificmolecules comprising at least one first binding specificity fordendritic cells and a second binding specificity for a second targetepitope. In a preferred embodiment of the invention, the second targetepitope is an antigen on a target cell, e.g. a tumor cell antigen, amicrobial antigen, a viral antigen or an autoantigen. These bispecificand multispecific molecules target dendritic cells to target cells suchthat the dendritic cells can modulate an immune response against such atarget cell or target cell antigen. In another embodiment of theinvention, the second target epitope is an Fc receptor, e.g., humanFcγRI (CD64) or a human Fcα receptor (CD89). Therefore, the inventionincludes bispecific and multispecific molecules capable of binding bothto FcγR, FcαR or FcεR expressing effector cells (e.g., monocytes,macrophages or polymorphonuclear cells (PMNs)), and to dendritic cells.These bispecific and multispecific molecules target dendritic cells toeffector cells and, like the human monoclonal antibodies of theinvention, may trigger Fc receptor-mediated effector cell activities,such as phagocytosis of dendritic cells, antibody dependentcell-mediated cytotoxicity (ADCC), cytokine release, or generation ofsuperoxide anion

Bispecific and multispecific molecules of the invention can furtherinclude a third binding specificity, in addition to an anti-Fc bindingspecificity or an anti-target cell antigen, and an anti-dendritic cellbinding specificity. In one embodiment, the third binding specificity isan anti-enhancement factor (EF) portion, e.g., a molecule which binds toa surface protein involved in cytotoxic activity and thereby increasesthe immune response against the target cell. The “anti-enhancementfactor portion” can be an antibody, functional antibody fragment or aligand that binds to a given molecule, e.g., an antigen or a receptor,and thereby results in an enhancement of the effect of the bindingdeterminants for the F_(C) receptor, target cell antigen or dendriticcell. The “anti-enhancement factor portion” can bind an F_(C) receptor,target cell antigen, or dendritic cell. Alternatively, theanti-enhancement factor portion can bind to an entity that is differentfrom the entity to which the first and second binding specificitiesbind. For example, the anti-enhancement factor portion can bind acytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1) orother immune cell that results in an increased immune response againstthe target cell.

In one embodiment, the bispecific and multispecific molecules of theinvention comprise as a binding specificity at least one antibody, or anantibody fragment thereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv,or a single chain Fv. The antibody may also be a light chain or heavychain dimer, or any minimal fragment thereof such as a Fv or a singlechain construct as described in Ladner et al. U.S. Pat. No. 4,946,778,issued Aug. 7, 1990, the contents of which is expressly incorporated byreference.

In another embodiment, bispecific and multispecific molecules of theinvention comprise a binding specificity for an antigen on a targetcell, e.g. a tumor cell antigen, a microbial antigen, a viral antigen oran autoantigen, and a second binding specificity for dendritic cells.

In another embodiment bispecific and multispecific molecules of theinvention comprise a binding specificity for an FcαR or an FcγR presenton the surface of an effector cell, and a second binding specificity fordendritic cells. The binding specificity for an Fc receptor can beprovided, for example, by another monoclonal antibody, including anotherhuman antibody. For example, the antibody can bind to an Fcγ receptor,preferably at a site which is not blocked by human immunoglobulin G(IgG). As used herein, the term “IgG receptor” refers to any of theeight γ-chain genes located on chromosome 1. These genes encode a totalof twelve transmembrane or soluble receptor isoforms which are groupedinto three Fcγ receptor classes: FcγRI (CD64), Fcγ RII (CD32), andFcγRIII (CD16). In one preferred embodiment, the Fcγ receptor a humanhigh affinity FcγRI. The human FcγRI is a 72 kDa molecule, which showshigh affinity for monomeric IgG (10⁸-10⁹M⁻¹).

The production and characterization of such anti-FcγR monoclonalantibodies are described by Fanger et al. in PCT application WO 88/00052and in U.S. Pat. No. 4,954,617, the teachings of which are fullyincorporated by reference herein. These antibodies bind to an epitope ofFcγRI, FcγRII or FcγRIII at a site which is distinct from the Fcγbinding site of the receptor and, thus, their binding is not blockedsubstantially by physiological levels of IgG. Specific anti-FcγRIantibodies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62and mAb 197. The hybridoma producing mAb 32 is available from theAmerican Type Culture Collection, ATCC Accession No. HB9469. Anti-FcγRImAb 22, F(ab′)₂ fragments of mAb 22, and can be obtained from Medarex,Inc. (Annandale, N.J.). In other embodiments, the anti-Fcγ receptorantibody is a humanized form of monoclonal antibody 22 (H22). Theproduction and characterization of the H22 antibody is described inGraziano, R. F. et al. (1995) J. Immunol 155 (10): 4996-5002 andPCT/US93/10384. The H22 antibody producing cell line was deposited atthe American Type Culture Collection on Nov. 4, 1992 under thedesignation HA022CL1 and has the accession no. CRL 11177.

In still another particular embodiment, the binding specificity for anFc receptor is provided by an antibody that binds to a human IgAreceptor, e.g., an Fc-alpha receptor (FcαRI (CD89)). The term “IgAreceptor” is intended to include the gene product of one α-gene (FcαRI)located on chromosome 19. This gene is known to encode severalalternatively spliced transmembrane isoforms of 55 to 110 kDa. FcαRI(CD89) is constitutively expressed on monocytes/macrophages,eosinophilic and neutrophilic granulocytes, but not on non-effector cellpopulations. FcαRI has medium affinity (≈5×10⁷M⁻¹) for both IgA1 andIgA2, which is increased upon exposure to cytokines such as G-CSF orGM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology16:423-440). Four FcαRI-specific monoclonal antibodies, identified asA3, A59, A62 and A77, which bind FcαRI outside the IgA ligand bindingdomain, have been described (Monteiro, R. C. et al., 1992, J. Immunol.148:1764).

FcαRI and FcγRI are preferred trigger receptors for use in the inventionbecause they are (1) expressed primarily on immune effector cells, e.g.,monocytes, PMNs, macrophages and dendritic cells; (2) expressed at highlevels (e.g., 5,000-100,000 per cell); (3) mediators of cytotoxicactivities (e.g., ADCC, phagocytosis); (4) mediate enhanced antigenpresentation of antigens, including self-antigens, targeted to them.

In all embodiments, bispecific and multispecific molecules of theinvention comprise at least one binding specificity which recognizes,e.g., binds to, dendritic cells

An “effector cell specific antibody” as used herein refers to anantibody or functional antibody fragment that binds the Fc receptor ofeffector cells. Preferred antibodies for use in the subject inventionbind the Fc receptor of effector cells at a site which is not bound byendogenous immunoglobulin.

As used herein, the term “effector cell” refers to an immune cell whichis involved in the effector phase of an immune response, as opposed tothe cognitive and activation phases of an immune response. Exemplaryimmune cells include a cell of a myeloid or lymphoid origin, e.g.,lymphocytes (e.g., B cells and T cells including cytolytic T cells(CTLs)), killer cells, natural killer cells, macrophages, monocytes,eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mastcells, and basophils. Some effector cells express specific Fc receptorsand carry out specific immune functions. In preferred embodiments, aneffector cell is capable of inducing antibody-dependent cell-mediatedcytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. Forexample, monocytes, macrophages, which express FcR are involved inspecific killing of target cells and presenting antigens to othercomponents of the immune system, or binding to cells that presentantigens. In other embodiments, an effector cell can phagocytose atarget antigen, target cell, or microorganism. The expression of aparticular FcR on an effector cell can be regulated by humoral factorssuch as cytokines. For example, expression of FcγRI has been found to beup-regulated by interferon gamma (IFN-γ). This enhanced expressionincreases the cytotoxic activity of FcγRI-bearing cells against targets.An effector cell can phagocytose or lyse a target antigen or a targetcell.

“Target cell” means any undesirable cell in a subject (e.g., a human oranimal) that can be targeted by a composition (e.g., a human monoclonalantibody, a bispecific or a multispecific molecule) of the invention. Inone embodiment, the target cell is a dendritic cell. In otherembodiments, a target cell includes a tumor cell, a microbial pathogen,a virus, or a virus infected cell.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific or multispecific molecules of theinvention are murine, chimeric and humanized monoclonal antibodies.

Chimeric mouse-human monoclonal antibodies (i.e., chimeric antibodies)can be produced by recombinant DNA techniques known in the art. Forexample, a gene encoding the Fc constant region of a murine (or otherspecies) monoclonal antibody molecule is digested with restrictionenzymes to remove the region encoding the murine Fc, and the equivalentportion of a gene encoding a human Fc constant region is substituted.(see Robinson et al., International Patent Publication PCT/US86/02269;Akira, et al., European Patent Application 184,187; Taniguchi, M.,European Patent Application 171,496; Morrison et al., European PatentApplication 173,494; Neuberger et al., International Application WO86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent Application 125,023; Better et al. (1988 Science240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987,J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimuraet al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

The chimeric antibody can be further humanized by replacing sequences ofthe Fv variable region which are not directly involved in antigenbinding with equivalent sequences from human Fv variable regions.General reviews of humanized chimeric antibodies are provided byMorrison, S. L., 1985, Science 229:1202-1207 and by Oi et al., 1986,BioTechniques 4:214. Those methods include isolating, manipulating, andexpressing the nucleic acid sequences that encode all or part ofimmunoglobulin Fv variable regions from at least one of a heavy or lightchain. Sources of such nucleic acid are well known to those skilled inthe art and, for example, may be obtained from 7E3, ananti-GPII_(b)III_(a) antibody producing hybridoma. The recombinant DNAencoding the chimeric antibody, or fragment thereof, can then be clonedinto an appropriate expression vector. Suitable humanized antibodies canalternatively be produced by CDR substitution U.S. Pat. No. 5,225,539;Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science239:1534; and Beidler et al. 1988 J. Immunol. 141:4053-4060.

All of the CDRs of a particular human antibody may be replaced with atleast a portion of a non-human CDR or only some of the CDRs may bereplaced with non-human CDRs. It is only necessary to replace the numberof CDRs required for binding of the humanized antibody to the Fcreceptor.

An antibody can be humanized by any method, which is capable ofreplacing at least a portion of a CDR of a human antibody with a CDRderived from a non-human antibody. Winter describes a method which maybe used to prepare the humanized antibodies of the present invention (UKPatent Application GB 2188638A, filed on Mar. 26, 1987), the contents ofwhich is expressly incorporated by reference. The human CDRs may bereplaced with non-human CDRs using oligonucleotide site-directedmutagenesis as described in International Application WO 94/10332entitled, Humanized Antibodies to Fc Receptors for Immunoglobulin G onHuman Mononuclear Phagocytes.

Also within the scope of the invention are chimeric and humanizedantibodies in which specific amino acids have been substituted, deletedor added. In particular, preferred humanized antibodies have amino acidsubstitutions in the framework region, such as to improve binding to theantigen. For example, in a humanized antibody having mouse CDRs, aminoacids located in the human framework region can be replaced with theamino acids located at the corresponding positions in the mouseantibody. Such substitutions are known to improve binding of humanizedantibodies to the antigen in some instances. Antibodies in which aminoacids have been added, deleted, or substituted are referred to herein asmodified antibodies or altered antibodies.

The term modified antibody is also intended to include antibodies, suchas monoclonal antibodies, chimeric antibodies, and humanized antibodieswhich have been modified by, e.g., deleting, adding, or substitutingportions of the antibody. For example, an antibody can be modified bydeleting the constant region and replacing it with a constant regionmeant to increase half-life, e.g., serum half-life, stability oraffinity of the antibody. Any modification is within the scope of theinvention so long as the bispecific and multispecific molecule has atleast one antigen binding region specific for an FcR and triggers atleast one effector function.

Bispecific and multispecific molecules of the present invention can bemade using a variety of known techniques including, but not limited to,chemical techniques (see e.g., D. M. Kranz et al. (1981) Proc. Natl.Acad. Sci. USA 78:5807), “polydoma” techniques (See U.S. Pat. No.4,474,893, to Reading), and recombinant DNA techniques.

In particular, bispecific and multispecific molecules of the presentinvention can be prepared by conjugating the constituent bindingspecificities, e.g., the anti-FcR and anti-dendritic cell bindingspecificities, using methods known in the art and described in theexamples provided herein. For example, each binding specificity of thebispecific and multispecific molecule can be generated separately andthen conjugated to one another. When the binding specificities areproteins or peptides, a variety of coupling or cross-linking agents canbe used for covalent conjugation. Examples of cross-linking agentsinclude protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate(SATA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB),o-phenylenedimaleimide (oPDM),N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described by Paulus (Behring Ins. Mitt. (1985) No.78, 118-132); Brennan et al. (Science (1985) 229:81-83), and Glennie etal. (J. Immunol. (1987) 139: 2367-2375). Preferred conjugating agentsare SATA and sulfo-SMCC, both available from Pierce Chemical Co.(Rockford, Ill.).

When the binding specificities are antibodies (e.g., two humanizedantibodies), they can be conjugated via sulfhydryl bonding of theC-terminus hinge regions of the two heavy chains. In a particularlypreferred embodiment, the hinge region is modified to contain an oddnumber of sulfhydryl residues, preferably one, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific and multispecific molecule is amAb×mAb, mAb×Fab, Fab×F(ab′)₂ or ligand×Fab fusion protein. A bispecificand multispecific molecule of the invention, e.g., a bispecific moleculecan be a single chain molecule, such as a single chain bispecificantibody, a single chain bispecific molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific andmultispecific molecules can also be single chain molecules or maycomprise at least two single chain molecules. Methods for preparing bi-and multspecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific and multispecific molecules to their specifictargets can be confirmed by enzyme-linked immunosorbent assay (ELISA), aradioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growthinhibition), or a Western Blot Assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a γ counter or a scintillationcounter or by autoradiography.

IV. Antibody Vaccine Conjugates/Immunotoxins

In another aspect, the present invention provides a variety oftherapeutic conjugates which include one or more human anti-dendriticcell antibodies (or fragments thereof) linked to a second agent.Particular agents which can be linked to the antibodies include, but arenot limited to, antigens (thereby forming a vaccine), radioisotopes,cytotoxins (thereby forming an immunotoxin) and other drugs. A cytotoxinor cytotoxic agent includes any agent that is detrimental to (e.g.,kills) cells. Examples include taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). An antibody of the presentinvention also can be conjugated to a radioisotope, e.g., radioactiveiodine, to generate cytotoxic radiopharmaceuticals for treating adendritic-related disorder, such as an autoimmune or inflammatorydisease, or graft versus host disease.

Accordingly, the antibody conjugates of the invention can be used tomodify a given biological response, and the drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, anenzymatically active toxin, or active fragment thereof, such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor or interferon; or, biological response modifierssuch as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moieties to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Human anti-dendritic cell antibodies of the invention (and fragmentsthereof) also can be linked to one or more antigens, such as a tumor orviral antigen, to form a vaccine conjugate. This allows for targeting ofa wide variety of antigens to dendritic cells to enhance processing,presentation and, ultimately, an immune response against the antigen(s).

Antibody-antigen vaccine conjugates of the invention can be madegenetically or chemically. In either case, the antibody portion of theconjugate may an consist of the whole antibody or a portion of theantibody, such as the Fab fragment or single-chain Fv. In addition, morethan one antigen can be added to a single antibody construct.

Genetically constructed anti-dendritic antibody-antigen conjugates(e.g., those expressed as a single recombinant fusion protein) can bemade by linking the antigen of choice to the antibody at a variety oflocations. Particular genetically produced conjugates (fusionconstructs) of the invention include, for example, the constructreferred to herein as MMV4, shown in FIG. 14. Fusion construct MMV4comprises human anti-dendritic cell antibody B11 fused to Pmel17, atumor-associated antigen. The nucleotide sequence encoding MMV4 is shownin SEQ ID NO:8.

As exemplified in the genetic fusion construct MMV4, the antigen (e.g.,Pmel17) can be fused to the end of the CH₃ domain of the human antibodyheavy chain. The antigen also can be fused at the hinged region of theantibody heavy chain in Fab-fusion constructs, or in sequence with thevariable light and heavy chains (V_(H) and V_(L)) in single chain fusionconstructs (ScFv constructs). Alternatively, the antigen can be fused tothe antibody light chain instead of the antibody heavy chain.

Chemically constructed antibody-antigen conjugates can be made using avariety of well known and readily available cross-linking reagents.These cross-linking reagents can be homofunctional or heterofunctionalcompounds, such as SPDP, SATA, SMCC, DTNB, that form covalent linkageswith different reactive amino acid or carbohydrate side chains on theanti-dendritic antibody and selected antigen.

Any antigen that can be cloned and expressed or purified can be selectedfor use in the antibody-antigen vaccine conjugates of the presentinvention. Techniques for obtaining such antigens are well-known in theart. For example, tumor-associated antigens can be directly purifiedfrom cancer cells and identified by physiochemical techniques such astandem mass spectrometry. Alternatively, tumor-specific T-cell clonescan be tested against antigen-negative cells that have acquired antigenby being transfected with plasmid DNA clones to isolate the cloneexpressing the antigen. Synthetic peptides can then be constructed toprecisely identify the antigenic site or epitope.

A significant advantage of the antibody-antigen conjugates of thepresent invention is their ability to rapidly elicit strong immuneresponses from vaccines to thereby improve the efficacy of vaccination.Accordingly, infectious disease antigens and tumor antigens againstwhich immune responses are protective or therapeutic can be conjugatedto human anti-dendritic cell antibodies of the invention, such asantibody B11, to form highly effective vaccines. Examples of infectiousdisease antigens include, but are not limited to, viral proteins,bacterial proteins and carbohydrates, fungal proteins and carbohydrates.

Antibody-antigen conjugates of the invention also can be used to improvethe efficacy of vaccination against infectious organisms and theirtoxins that may be encountered during travel or through biowarfare.Examples of such antigens include, for example, anthrax antigens,botulism toxin, malaria antigens, equine encephalitis, and Y. pestisantigens.

Other suitable antigens for use in the antibody-antigen conjugates ofthe invention include tumor-associated antigens for the prevention ortreatment of cancers. Examples of tumor-associated antigens include, butare not limited to, HER2/neu, CEA, gp100, MART1, TRP-2, melan-A,NY-ESO-1, MN (gp250), idiotype, MAGE-1, MAGE-3, Tyrosinase, Telomerase,and MUC-1 antigens. Tumor associated antigens also include the bloodgroup antigens, for example, Le^(a), Le^(b), LeX, LeY, H-2, B-1, B-2antigens. In another preferred embodiment, more than one antigen isfused to a single anti-dendritic antibody construct. For example, a MAGEantigen can be combined with other antigens such as melanin A,tyrosinase, and gp100 along with adjuvants such as GM-CSF or IL-12, andfused to an anti-dendritic antibody construct, e.g., B11.

Other suitable antigens include viral antigens for the prevention ortreatment of viral diseases. Examples of viral antigens include, but arenot limited to, HIV-1 gag, HIV-1 env, HIV-1 nef, HBV core, FAS, HSV-1,HSV-2, p17, ORF2 and ORF3 antigens. In another preferred embodiment, theselected antigen is a melanoma-specific antigen including, but notlimited to, gp100 or Pmel17. In another preferred embodiment, theselected antigen is a prostate-specific cancer antigen, for example,PSMA. In yet another embodiment, the selected antigen is a bacterialantigen including, but not limited to, Toxoplasma gondii or Treponemapallidum. The antibody-bacterial antigen conjugates of the invention canbe in the treatment or prevention of various bacterial diseases such asAnthrax, Botulism, Tetanus, Chlamydia, Cholera, Diptheria, Lyme Disease,Syphilis and Tuberculosis.

In another aspect, human antibodies specific for dendritic cells can beused to directly target whole cells, e.g., a tumor cell, an effectorcell or a microbial pathogen, to dendritic cells. Anti-dendritic cellantibodies or antigen binding fragments thereof can be directlyexpressed on the surface of a cell, for example, by transfection ortransduction of a cell with a vector containing nucleic acid sequencesencoding a human dendritic cell-specific antibody of the invention, orantigen binding fragment thereof. This can be done, for example, bytransfecting the target cell with a nucleic acid encoding a fusionprotein containing a transmembrane domain and a human anti-dendriticcell antibody, or antigen binding fragment thereof. Methods forgenerating such nucleic acids, fusion proteins, and cells expressingsuch fusion proteins are described, for example, in U.S. patentapplication Ser. No. 09/203,958, incorporated herein in its entirety bythis reference. Alternatively, anti-dendritic cell antibodies, orantigen binding fragments thereof, can be bound to a cell or a pathogenby the use of chemical linkers, lipid tags, or other related methods(deKruif, J. et al. (2000) Nat. Med. 6:223-227; Nizard, P. et al. (1998)FEBS Lett. 433:83-88). Cells with surface-anchored anti-dendritic cellantibodies, or an antigen binding fragments thereof, may be used toinduce specific immune responses against the cell, e.g., a tumor cell ormicrobial pathogen.

V. Pharmaceutical Compositions

In another aspect, the present invention provides therapeuticcompositions, e.g., pharmaceutical compositions, containing one or acombination of human monoclonal antibodies, or antigen-bindingportion(s) thereof, of the present invention, formulated together with apharmaceutically acceptable carrier. Such compositions can additionallyinclude other therapeutic reagents, such as other antibodies, cytotoxinsor drugs (e.g., immunosuppressants), and can be administered alone or incombination with other therapies, such as radiation.

In one embodiment, human anti-dendritic cell monoclonal antibodieshaving complementary activities are used in combination, e.g., as apharmaceutical composition, comprising two or more human anti-dendriticmonoclonal antibodies. For example, a human monoclonal antibody thatmediates highly effective killing of dendritic cells in the presence ofeffector cells can be combined with another human monoclonal antibodythat inhibits the growth of dendritic cells. In another embodiment, ahuman monoclonal antibody that is rapidly internalized by dendriticcells can be combined with another human monoclonal antibody thatinduces antigen presenting cell activities of dendritic cells, e.g.,release of immunostimulatory cytokines.

In another embodiment, the composition comprises one or a combination ofbispecific or multispecific molecules of the invention (e.g., whichcontains at least one binding specificity for an Fc receptor and atleast one binding specificity for dendritic cells).

In yet another embodiment, the composition comprises at least onebinding specificity for dendritic cells functionally linked to anothermolecular entity, for example, a cytotoxin, or an antigen, e.g., anantigen on a target cell.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,bispecific and multispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

To administer a compound of the invention by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present inventioninclude those suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the subject beingtreated, and the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compositionwhich produces a therapeutic effect. Generally, out of one hundredpercent, this amount will range from about 0.01 percent to aboutninety-nine percent of active ingredient, preferably from about 0.1percent to about 70 percent, most preferably from about 1 percent toabout 30 percent.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate. Dosage forms for the topical or transdermaladministration of compositions of this invention include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given alone or as apharmaceutical composition containing, for example, 0.01 to 99.5% (morepreferably, 0.1 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitabledaily dose of a compositions of the invention will be that amount of thecompound which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. It is preferred that administration be intravenous,intramuscular, intraperitoneal, or subcutaneous, preferably administeredproximal to the site of the target. If desired, the effective daily doseof a therapeutic compositions may be administered as two, three, four,five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms. While itis possible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation (composition).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example,the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134),different species of which may comprise the formulations of theinventions, as well as components of the invented molecules; p120(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273. In one embodiment of the invention, thetherapeutic compounds of the invention are formulated in liposomes; in amore preferred embodiment, the liposomes include a targeting moiety. Ina most preferred embodiment, the therapeutic compounds in the liposomesare delivered by bolus injection to a site proximal to the tumor orinfection. The composition must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi.

A “therapeutically effective dosage” preferably modulates dendritic cellgrowth and/or activity by at least about 20%, more preferably by atleast about 40%, even more preferably by at least about 60%, and stillmore preferably by at least about 80% relative to untreated subjects.The ability of a compound to modulate dendritic cell growth and/oractivity can be evaluated in an animal model system predictive ofefficacy in antigen presentation and/or immunomodulation.

Alternatively, this property of a composition can be evaluated byexamining the ability of the compound to modulate immune cellstimulation by dendritic cells, such as in in vitro by assays describedherein and known to the skilled practitioner. In one embodiment, atherapeutically effective amount of a therapeutic compound can inhibitdendritic cell growth and/or activity, or otherwise ameliorate symptoms,e.g., symptoms of autoimmunity, in a subject. In another embodiment, atherapeutically effective amount of a therapeutic compound can enhanceantigen processing and presentation by dendritic cells, and thus enhanceimmune responses against a immunogen or target antigen. One of ordinaryskill in the art would be able to determine such amounts based on suchfactors as the subject's size, the severity of the subject's symptoms,immune activity in the subject, and the particular composition or routeof administration selected.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carriercan be an isotonic buffered saline solution, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as manitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

When the active compound is suitably protected, as described above, thecompound may be orally administered, for example, with an inert diluentor an assimilable edible carrier.

VI. Uses and Methods of the Invention

The compositions (e.g., human monoclonal antibodies to dendritic cellsand derivatives/conjugates thereof) of the present invention have invitro and in vivo diagnostic and therapeutic utilities.

For example, these molecules can be administered to cells in culture,e.g. in vitro or ex vivo, or in a subject, e.g., in vivo, to treat,prevent or diagnose a variety of disorders. As used herein, the term“subject” is intended to include human and non-human animals. The term“non-human animals” of the invention includes all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dog, cow,chickens, amphibians, reptiles, etc.

In a particular embodiment, the human antibodies and derivatives thereofare used in vivo to treat, prevent or diagnose a variety of dendriticcell-mediated or dendritic cell-related diseases.

In one embodiment, preferred human animals include a human patienthaving dendritic cell-mediated or a dendritic cell-related disease. Forexample, the methods and compositions of the present invention can beused to treat a subject with an autoimmune, immune system, orinflammatory disorder, e.g., a disorder characterized by aberrant orunwanted immune activity associated with immunomodulation by dendriticcells. Autoimmune, immune system, and inflammatory disorders that maybenefit from treatment with the human anti-dendritic cells of theinvention include rheumatoid arthritis, multiple sclerosis, diabetesmellitus, myasthenia gravis, pernicious anemia, Addison's disease, lupuserythematosus, Reiter's syndrome, and Graves disease. For example, asubject suffering from an autoimmune disorder may benefit frominhibition of dendritic cell mediated presentation of an autoantigen.

Other examples of diseases that can be treated using the humananti-dendritic cell antibodies of the invention include transplantrejection and graft versus host disease.

Transplant Rejection

Over recent years there has been a considerable improvement in theefficiency of surgical techniques for transplanting tissues and organssuch as skin, kidney, liver, heart, lung, pancreas and bone marrow.Perhaps the principal outstanding problem is the lack of satisfactoryagents for inducing immune-tolerance in the recipient to thetransplanted allograft or organ. When allogeneic cells or organs aretransplanted into a host (i.e., the donor and donee are differentindividual from the same species), the host immune system is likely tomount an immune response to foreign antigens in the transplant(host-versus-graft disease) leading to destruction of the transplantedtissue. CD8+ cells, CD4+ cells and monocytes are all involved in therejection of transplant tissues. The therapeutic agents of the presentinvention are useful to inhibit dendritic cell mediatedalloantigen-induced immune responses in the donee thereby preventingsuch cells from participating in the destruction of the transplantedtissue or organ.

Graft Versus Host Disease

A related use for the therapeutic agents of the present invention is inmodulating the immune response involved in “graft versus host” disease(GVHD). GVHD is a potentially fatal disease that occurs whenimmunologically competent cells are transferred to an allogeneicrecipient. In this situation, the donor's immunocompetent cells mayattack tissues in the recipient. Tissues of the skin, gut epithelia andliver are frequent targets and may be destroyed during the course ofGVHD. The disease presents an especially severe problem when immunetissue is being transplanted, such as in bone marrow transplantation;but less severe GVHD has also been reported in other cases as well,including heart and liver transplants. The therapeutic agents of thepresent invention are used to inhibit the activity of host antigenpresenting cells, e.g., dendritic cells.

In another embodiment, the methods and compositions of the invention canbe used to modulate an immune response in a subject towards an antigen.The human anti-dendritic cell antibodies of the invention can be used totarget an antigen to a dendritic cell and thereby modulate antigenpresentation and processing, such that an immune response to the antigenis induced. The antigen can be a tumor antigen, or an antigen from apathogen, e.g., a microbial pathogen. The pathogen can be a virus (e.g.,HIV), a bacterium, a fungus, or a parasite. The antigen can also be acomponent of an amyloid deposit in a patient, such as a patientsuffering from Alzheimer's disease and the antigen is Aβ peptide.

For example, a molecular complex comprising at least one bindingspecificity for a component on the surface of a dendritic cell linked toan antigen, wherein binding of the complex to the dendritic cellmediates internalization of the molecular complex, can be administeredto a subject to induce or enhance an immune response against theantigen. The immune response generated against the antigen includesantibodies that bind to the antigen and T cells that bind to the antigenas a component of an MHC-I or MHC-II complex. Accordingly, the humananti-dendritic cell antibodies of the invention can also be used tomediate dendritic cell-targeted immunization of a subject. For example,a subject can be immunized with a molecular complex comprising at leastone binding specificity for a component on the surface of a dendriticcell linked to an antigen, wherein binding of the complex to thedendritic cell mediates internalization of the molecular complex, and,for example, enhances processing and presentation of the antigen.

In another aspect, human antibodies specific for dendritic cells can beused to directly target whole cells, e.g., a tumor cell, an effectorcell or a microbial pathogen, to dendritic cells. Anti-dendritic cellantibodies or antigen binding fragments thereof can be directlyexpressed on the surface of a cell, for example, by transfection ortransduction of a cell with a vector containing nucleic acid sequencesencoding a human dendritic cell-specific antibody of the invention.Alternatively, anti-dendritic cell antibodies, or antigen bindingfragments thereof, can be bound to a cell or a pathogen by the use ofchemical linkers, or lipid tags, or other related methods. Cells withsurface-anchored anti-dendritic cell antibodies, or an antigen bindingfragments thereof, may be used to induce specific immune responsesagainst the cell, e.g., a tumor cell or microbial pathogen.

Thus, the antibodies of the invention can be used to stimulate theimmune response to pathogens, toxins, and self-antigens. Examples ofpathogens for which this therapeutic approach may be particularlyuseful, include pathogens for which there is currently no effectivevaccine, or pathogens for which conventional vaccines are less thancompletely effective. These include, but are not limited to HIV,Hepatitis (A, B, & C), Influenza, Herpes, Giardia, Malaria, Leishmania,Staphylococcus Aureus, Pseudomonas aeruginosa.

The compositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention can be initially tested for binding activityassociated with therapeutic or diagnostic use in vitro. For example,compositions of the invention can be tested using the flow cytometricand internalization assays described in the Examples below. Moreover,the activity of these molecules in triggering at least oneeffector-mediated effector cell activity, including cytolysis ofdendritic cells can be assayed. Protocols for assaying for effectorcell-mediated phagocytosis and cytolysis are known in the art.

The compositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention have additional utility in therapy anddiagnosis of dendritic cell-mediated or dendritic cell-related diseases.For example, the human monoclonal antibodies, the multispecific orbispecific molecules can be used, for example, to elicit in vivo or invitro one or more of the following biological activities: to opsonize adendritic cell; to mediate phagocytosis or cytolysis of a dendritic cellin the presence of human effector cells; to inhibit the growth of adendritic cell; to be internalized by a dendritic cell; or to target anantigen to a dendritic cell.

Methods of administering the compositions (e.g., human antibodies,multispecific and bispecific molecules) of the invention are known inthe art. Suitable dosages of the molecules used will depend on the ageand weight of the subject and the particular drug used. The moleculescan be coupled to radionuclides, such as ^(1□1)I, ⁹⁰Y, ¹⁰⁵Rh, etc., asdescribed in Goldenberg, D. M. et al. (1981) Cancer Res. 41: 4354-4360,and in EP 0365 997. The compositions (e.g., human antibodies,multispecific and bispecific molecules) of the invention can also becoupled to immunomodulatory agents.

Target-specific effector cells, e.g., effector cells linked tocompositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention can also be used as therapeutic agents.Effector cells for targeting can be human leukocytes such asmacrophages, neutrophils or monocytes. Other cells include eosinophils,natural killer cells and other IgG- or IgA-receptor bearing cells. Ifdesired, effector cells can be obtained from the subject to be treated.The target-specific effector cells, can be administered as a suspensionof cells in a physiologically acceptable solution. The number of cellsadministered can be in the order of 10⁸-10⁹ but will vary depending onthe therapeutic purpose. In one embodiment, the amount will besufficient to obtain localization at, for example, a dendritic cell, andto effect cell killing by, e.g., phagocytosis. In another embodiment,target-specific dendritic cells, e.g., dendritic cells linked tocompositions of the invention can be used as therapeutic agents forlocalization at a target cell, e.g., a tumor cell, microbial pathogen,virus, or virus infected cell, or for targeting an antigen, and toeffect an immune response against the target cell or a target antigen,by, e.g., antigen processing and presentation. Routes of administrationcan also vary.

Therapy with target-specific effector cells or target-specific dendriticcells can be performed in conjunction with other techniques for removalof targeted cells. For example, anti-dendritic cell therapy using thecompositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention and/or effector cells armed with thesecompositions can be used in conjunction with chemotherapy orimmunomodulatory therapy, e.g., anti-inflammatory or immunosuppressivetherapy. Additionally, combination immunotherapy may be used to directtwo distinct cytotoxic effector populations toward, for example,dendritic cells. For example, anti-dendritic cell antibodies linked toanti-Fc-gammaRI or anti-CD3 may be used in conjunction with IgG- orIgA-receptor specific binding agents.

In another embodiment, dendritic cells targeted with, for example, humanantibodies, multispecific and bispecific molecules of the invention, canbe used in conjunction with immunomodulatory therapy, e.g.,immunstimulation, to enhance an immune response against a target cell ora target antigen. A dendritic cell targeted therapy can be combined withother forms of immunotherapy such as cytokine treatment (e.g.interferons, TNFα, GM-CSF, G-CSF, IL-2).

Bispecific and multispecific molecules of the invention can also be usedto modulate dendritic cell activities, e.g., antigen processing andpresentation, as well as to modulate the level of a cognate antigen on adendritic cell, such as by capping and elimination of receptors on thecell surface.

The compositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention which have complement binding sites, such asportions from IgG1, -2, or -3 or IgM which bind complement, can also beused in the presence of complement. In one embodiment, ex vivo treatmentof a population of cells comprising target cells with a binding agent ofthe invention and appropriate effector cells can be supplemented by theaddition of complement or serum containing complement. Phagocytosis oftarget cells coated with a binding agent of the invention can beimproved by binding of complement proteins. In another embodiment targetcells coated with the compositions (e.g., human antibodies,multispecific and bispecific molecules) of the invention can also belysed by complement.

The compositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention can also be administered together withcomplement. Accordingly, within the scope of the invention arecompositions comprising human antibodies, multispecific or bispecificmolecules and serum or complement. These compositions are advantageousin that the complement is located in close proximity to the humanantibodies, multispecific or bispecific molecules. Alternatively, thehuman antibodies, multispecific or bispecific molecules of the inventionand the complement or serum can be administered separately.

In other embodiments, the subject can be additionally treated with anagent that modulates, e.g., enhances or inhibits, immune cell activityand/or the expression or activity of Fcα or Fcγ receptors, by forexample, treating the subject with a cytokine. Preferred cytokines foradministration during treatment with the multispecific molecule includeof granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumornecrosis factor (TNFα).

The compositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention can also be used to target, for example,dendritic cells, e.g., for labeling such cells. For such use, thebinding agent can be linked to a molecule that can be detected. Thus,the invention provides methods for localizing ex vivo or in vitrodendritic cells. The detectable label can be, e.g., a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor.

In one embodiment, the invention provides methods for detecting thepresence of dendritic cells or a dendritic cell antigen in a sample, ormeasuring the amount of dendritic cells or a dendritic cell antigen,comprising contacting the sample, and a control sample, with a humanmonoclonal antibody, or an antigen binding portion thereof, whichspecifically binds to dendritic cells or a dendritic cell antigen, underconditions that allow for formation of a complex between the antibody orportion thereof and dendritic cells or a dendritic cell antigen. Theformation of a complex is then detected, wherein a difference complexformation between the sample compared to the control sample isindicative the presence of dendritic cells or a dendritic cell antigenin the sample.

In still another embodiment, the invention provides a method fordetecting the presence or quantifying the amount of dendritic cells invivo or in vitro. The method comprises (i) administering to a subject acomposition (e.g., a multi- or bispecific molecule) of the invention ora fragment thereof, conjugated to a detectable marker; (ii) exposing thesubject to a means for detecting said detectable marker to identifyareas containing dendritic cells.

Also within the scope of the invention are kits comprising thecompositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention and instructions for use. The kit canfurther contain a least one additional reagent, such as a cytokine orcomplement, or one or more additional human antibodies of the invention(e.g., a human antibody having a complementary activity which binds toan epitope on dendritic cells distinct from the first human antibody).

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Example 1 Production of Human Monoclonal Antibodies AgainstDendritic Cells

Human anti-dendritic cell monoclonal antibodies were generated byimmunizing the HCO7 strain of HuMAb mice with preparations of dendriticcells. HCO7 HuMAb mice were generated as described in U.S. Pat. Nos.5,770,429 and 5,545,806, the entire disclosures of which are herebyincorporated by reference.

In particular, HCO7 mice were immunized four times with intraperitonealinjections of human dendritic cells emulsified in Freund's Adjuvant.Briefly, dendritic cells were prepared as follows. Human peripheralblood mononuclear cells (PBMCs) were obtained by density gradientcentrifugation of whole blood or Leukopak platelet apheresispreparations. Monocytes were isolated by adherance to tissue cultureflasks for two hours, and then differentiated into dendritic cells byincubation with 2 ng/ml GM-CSF and 10 ng/ml IL-4 in macrophage serumfree media (Gibco) for 5 to 9 days. Cells for immunizations were usedfresh or stored frozen at −80° C. Mice were immunized every 2-3 weeks.Finally, an intravenous injection of dendritic cells in phosphatebuffered saline (PBS) was performed prior to splenectomy. The spleensfrom responding mice were harvested and dispersed into single cells.

To generate hybridomas producing anti-dendritic cell antibodies,splenocytes from mice with plasma containing anti-dendritic cellantibodies were fused with P3X63-Ag8.653 myeloma cells (deposited withthe ATCC under designation ATCC CRL 1580 nonsecreting mouse myelomacells) and PEG. Hybridomas were selected by growth in HAT containingmedia. After hybridomas grew out (about 10-14 days) each well containinghybridomas was screened for the production of human IgG using ananti-human IgG ELISA.

Positive hybridomas were screened for and selected based on thefollowing properties: (1) production of human IgG antibodies, and (2)binding to dendritic cells.

The hybridomas secreting human IgG were tested for reactivity withvarious types of blood cells by flow cytometry. Dendritic cells wereprepared from adherent mononuclear cells by culturing for 5-7 days inmedia supplemented with GM-CSF and IL-4. Granulocytes (PMN), monocytesand lymphocytes were obtained from heparanized whole blood. The cellswere incubated with hybridoma supernatants from IgG-positive clones at4° C. Binding was detected with a FITC-labeled goat anti-human IgG(Fc)probe. The cell associated fluorescence was determined by analysis usinga FACScalibur instrument.

Several hybridomas that were screened produced human IgG1κ antibodiesthat demonstrated reactivity with dendritic cells as assessed by flowcytometry (e.g., A3, A5, A23, A24, A33, B9, B11, B33, B47, C8, C10, C20,C28, C29, C30, C35, E1, E8, E10, E18, E20, E21, E24, 10F7, 28-6, 24-3,25-14, 17-6, 19-2 and 19-4), as shown in Table 1 below. Some of thehuman antibodies demonstrated very high and preferential reactivity withdendritic cells as compared to other blood cell types.

TABLE 1 Human Monoclonal Antibodies with Reactivity to Dendritic CellsHuman MAb Lymphocytes Monocytes PMNs Dendritic Cells A3 − +/− +/− + A5 −+/− − +/− A23 − + − +/− A24 − ++ + ++ A33 − +/− − +/− B9 +/− +++ + +++B11 − +/− − +++ B33 − +/− − +/− B47 − +/− +/− +/− C8 − +/− − +/− C10 −+/− +/− + C20 − +/− +/− ++ C28 − +/− − +/− C29 − +/− +/− ++ C30 − − −+/− C35 − +/− − ++ E1 − +/− − + E8 − + + ++ E10 − + + +++ E18 − + + ++E20 + ++ +/− +++ E21 +/− ++ +/− +++ E24 − +/− − +/− 10F7 − +/− + ++28-6 + + + ++ 24-3 + − − ++ 25-14 − + − ++ 17-6 − + − + 19-2 − − − +19-4 + − − + Key: − no binding detected +/− weak/equivocal binding +low/significant binding ++ high binding +++ extremely high binding

Example 2 Characterization of Human Monoclonal Antibodies AgainstDendritic Cells I. Binding Specificity of Purified Human Anti-DendriticCell Antibodies to Dendritic Cells

Several hybridomas that secreted human IgG antibodies with specificityfor dendritic cells were subcloned and expanded for purification.Monoclonal antibodies were isolated from supernatants of hybridomacultures grown in spinner flasks in a humidified incubator containing 5%CO2. Antibodies were purified by chromatography on a Protein A-agarosecolumn according to the manufacturer's specifications (Pierce, RockfordIll.).

The purified human antibodies were then tested for reactivity withdendritic cells and cell lines representing various other hematopoeticcell types using flow cytometry. Briefly, dendritic cells were preparedfrom adherent mononuclear cells by culturing for 5-7 days in mediasupplemented with GM-CSF and IL-4, as described above. The U937, CEM,THP-1 and L540 cell lines were cultured in media supplemented with 10%fetal bovine serum. The cells were harvested, washed, and incubated withsaturating concentrations of human monoclonal antibodies B11, C20, E21or an isotype control (human IgG1) at 4° C. with shaking for 1 hour.Antibody binding was detected by further incubation with a FITC-labeledgoat anti-human IgG(Fc) probe for 1 hour at 4° C. The cells were washed,fixed with 1% paraformaldehyde, and cell associated fluorescence wasanalyzed using a FACScalibur (Beckton Dickinson) instrument withCellQuest software.

As shown in FIG. 1, human monoclonal antibody B11 bound exclusively todendritic cells. Monoclonal antibody C20 bound specifically to dendriticcells, and also demonstrated low level reactivity with the monocyte-likecell lines U-937 and THP-1, and the Hodgkin's lymphoma cell line L540.Similarly, human monoclonal antibody E21 bound preferentially todendritic cells, but also reacted at a low level with L540 cells andTHP-1 cells. These data demonstrate that human monoclonal antibodiesB11, C20 and E21 recognize different antigens, and that theypreferentially bind to dendritic cells compared to other cells ofhematopoetic lineage.

II. Dose-Dependent Binding of Purified Human Anti-Dendritic CellAntibodies to Dendritic Cells

The dose-dependent reactivity of purified human anti-dendritic cellmonoclonal antibodies B11, C20, and E21 with dendritic cells wasexamined by flow cytometry. Dendritic cells were prepared from adherentmononuclear cells as described above. The cells were harvested andincubated with varying concentrations of the monoclonal antibodies B11,C20, E21 or an isotype control at 4° C. Antibody binding was detectedwith a FITC-labeled goat anti-human IgG(Fc) probe, and cell associatedfluorescence was determined using a FACScalibur instrument withCellQuest software.

Each monoclonal antibody demonstrated dose-dependent binding todendritic cells as compared to an isotype matched control IgG antibody,as shown in FIG. 2. These data demonstrate that the purified humanmonoclonal antibodies B11, C20 and E2 bind in a concentration-dependentmanner to dendritic cells. The varying intensity of binding between theanti-dendritic cell antibodies indicates that they recognize uniquemolecules or epitopes on the dendritic cells.

III. Binding of Human Antibody B11 to CD34+ Stem Cell-Derived dendriticCells

Due to their availability, dendritic cells differentiated fromcirculating blood monocytes are the most commonly used type of dendriticcell for both research and clinical applications. However, dendriticcells derived from progenitor stem cells also can be used and may moreaccurately represent dendritic cells in human tissues. Accordingly, inthe following study, dendritic cells differentiated from CD34+progenitor cells were evaluated for reactivity with human monoclonalantibody B11 by flow cytometry.

Purified monoclonal antibody B11 was extensively dialyzed against 0.3 Msodium carbonate buffer, pH 9.5, for labeling with fluorescenceisothiocyanate (FITC). A stock FITC solution was prepared by dissolving1 mg solid FITC in 1 ml of DMSO. Stock FITC was added dropwise withconstant mixing in an amount to provide 50 μg FITC per mg of antibodyprotein. Following the addition of FITC, the solution was incubated inthe dark for 1-3 hours at room temperature. FITC labeled antibody wasisolated by gel filtration on a Sephadex G-10 column equilibrated inPBS.

CD34+ progenitor cells and dendritic cell differentiation media wereobtained from Poetic Technologies, Inc. (Gaithersburg, Md.). The cellswere differentiated according to the manufacturer's instructions.Dendritic cells were harvested and incubated with varying concentrationsof B11-FITC or an isotype control antibody (human IgG-FITC) at 4° C. Thecell associated fluorescence was determined by analysis using aFACScalibur instrument with CellQuest software.

The results are shown in FIG. 3 and demonstrate that human monoclonalantibody B11 binds to dendritic cells differentiated from CD34+ stemcells in a dose dependent manner. Therefore, the B11 target antigen isexpressed on dendritic cells that are derived from monocytes and fromprogenitor stem cells.

IV. Binding of Human Antibody B11 to Macrophages and Dendritic Cells

The ability of human anti-dendritic cell monoclonal antibody B11 to bindto macrophages compared dendritic cells was assessed by flow cytometry.

Dendritic cells were prepared from adherent mononuclear cells asdescribed above. Macrophages were prepared from adherent mononuclearcells by culturing for 5-7 days with M-CSF. The cells were harvested andincubated with 10 ug/ml of monoclonal antibody B11 or an isotype controlantibody at 4° C. Human antibody binding was detected with aFITC-labeled goat anti-human IgG(Fc) probe. The cell associatedfluorescence was determined by analysis using a FACScalibur instrumentwith CellQuest software.

The results are shown in FIG. 4 and demonstrate that human antibody B11binds to macrophages to a lesser extent than to dendritic cells. Thus,the B11 target antigen is also expressed on macrophages, although thelevel of expression is lower than that observed on dendritic cells. Thereactivity of monoclonal antibody B11 with macrophages is not surprisingdue to the similarity between dendritic cells and macrophages. Sincethese two cell types share both structural and functional properties,including the ability to stimulate T and B lymphocyte responses, thecross-reactivity of antibody B11 with macrophages may be beneficial fortargeting antigen presenting cells.

V. Induction of Human Antibody B11 Target Antigen on THP-1 Cells

Human monoclonal antibody B11 was tested using flow cytometry forbinding to THP-1 cells, a monocyte-like cell line derived from a humanmonocytic leukemia, before and after the cells had been induced todifferentiate towards a dendritic cell phenotype.

Briefly, THP-1 cells were grown in standard culture media or in mediasupplemented with GM-CSF and IL-4. The cells were incubated with 10μg/ml of monoclonal antibody B11-FITC or an isotype control antibody(human IgG-FITC) at 4° C. The cell associated fluorescence wasdetermined by analysis using a FACScalibur instrument with CellQuestsoftware. The results are shown in FIG. 5.

These data demonstrate that, under normal growth conditions, THP-1 cellsdo not express the B11 target antigen. However, when THP-1 cells aredriven towards a dendritic cell phenotype by culturing in mediacontaining GM-CSF and IL-4, the expression of the B11 target antigen isconcomitantly induced. Accordingly, these results further confirm thespecificity of the B11 human antibody for a target antigen (B11)associated specifically with dendritic cells.

VI. Binding of Human Antibody B11 to Macaque Dendritic Cells

The animal (monkey) model of cynomolgus macaques can provide relevantinformation regarding the clinical application of antibodies, providedthat the target antigen is conserved among primates. Accordingly, thecross-reactivity of human monoclonal antibody B11 with dendritic cellsfrom cynomolgus monkey was evaluated by flow cytometry.

Fresh cynomolgus blood was obtained from Sierra Biomedicals, anddendritic cells were prepared from adherent mononuclear cells byculturing with GM-CSF and IL-4. Dendritic cells were incubated with 10μg/ml of monoclonal antibody B11-FITC or an isotype control antibody(human IgG-FITC) at 4° C. The cell associated fluorescence wasdetermined by analysis using a FACScalibur instrument.

As shown in FIG. 6, human monoclonal antibody B11 binds to dendriticcells derived from cynomolgus macaques, suggesting that the B11 targetantigen is conserved in primates.

VII. Binding of Human Antibody B11 to Dendritic Cells in Human Tissues

The reactivity of human monoclonal antibody B11 with dendritic cellsfrom human tissues was evaluated by immunohistochemistry. Theseexperiments were also designed to evaluate any potentialcross-reactivity of antibody B11 with other cells or antigens of humantissues.

Cryosections of human tissues were obtained via autopsy or surgicalbiopsy and embedded in Tissue-Tek O.C.T. medium and stored frozen below−70° C. Tissues were sectioned at 5 mm, fixed for 10 minutes withacetone, and dried overnight. Slides were fixed with 10%neutral-buffered formalin for 10 seconds prior to staining. An indirectimmunoperoxidase technique was employed. Sections were first stainedwith the B11-FITC or isotype-matched-FITC antibody diluted in PBScontaining heat aggregated IgG to block Fc-dependent binding. Primaryantibodies were detected using a rabbit anti-FITC antibody followed by aperoxidase labeled anti-rabbit reagent. Each slide was read by acertified pathologist and binding was rated according to the followingkey: ±(equivocal), 1+ (weak), 2+ (moderate), 3+ (strong), 4+ (intense),Neg. (negative). The results shown in Table 2 below demonstrate clearstaining of dendritic cells and some macrophages in all tissuesexamined. No specific staining was observed with the isotype controlantibody.

These data demonstrate that human monoclonal antibody B11 binds todendritic cells in human tissues, as well as macrophages in humantissues albeit to a lesser extent. The minimal binding of B11 tomononuclear cells in the spleen and progenitor cells of the bone marrowmay represent binding to immature dendritic cells. Human antibody B11did not cross-react with other cell types or tissues in the samplestested, further demonstrating the specificity of this antibody fordendritic cells and, to a lesser extent, macrophages.

TABLE 2 Immunohistochemistry of Human Monoclonal Antibody B11 Binding toHuman Tissues Antibody Tissue and reactivity B11-FITC (2 μg/ml) Skin:Dermal dendritic cells 3+, all other elements negative. ″ Tonsil:Interstitial and/or subepithelial dendritic cells 2+, all other elementsnegative. ″ Liver: Interstitial dendritic cells 2+, Kupffer cells 2+,all other elements negative. ″ Breast: Dermal/subcutaneous/interstitialdendritic cells 3-4+, all other elements negative. ″ Spleen:Interstitial dendritic cells 3-4+, Reticulotendothelial cells liningcords of Billiroth 2+, occasional mononuclear cells in marginal zone 2+,rare to occasional mononuclear cells in PALS/follicles 2+, otherelements negative. ″ Kidney: Interstitial dendritic cells 3-4+, allother elements negative. ″ Lymph node: Capsular dendritic cells 3-4+,subcapsular dendritic cells/macrophages 3+, follicular dendritic cells2-3+, paracortical dendritic cells 2-3+, medullary sinus dendriticcells/macrophages 1-2+, other elements negative. ″ Brain:Meningeal/peritheleal dendritic cells 3-4+, all other elements negative.″ Testis: Interstitial dendritic cells 3-4+, all other elementsnegative. ″ Pancreas: Interstitial dendritic cells 3-4+, all otherelements negative. ″ Heart: Interstitial dendritic cells 3-4+, all otherelements negative. ″ Small intestine: Interstitial dendritic cells 3-4+,Lamina propria dendritic cells/macrophages 2-3+, Peyers patch dendriticcells/macrophages 2-3+, other elements negative. ″ Bone Marrow:Interstitial dendritic cells 3-4+, Hematopoetic progenitors 2+, otherelements negative. ″ Lung: Interstitial dendritic cells 3-4+, Alveolarmacrophages 3-4+, other elements negative. IgG1-FITC (2 μg/ml) Alltested tissues: All elements negative.Tissue cross-reactivity studies were conducted at Pathology AssociatesInternational, Frederick, Md. study #IM598.

VIII. Binding of Single Chain Fv (ScFv) Fragments of Human Antibody B11to Human Dendritic Cells.

The reactivity of a single chain Fv fragment of human monoclonalantibody B11 with dendritic cells from human tissues was evaluated byflow cytometry.

The B11 ScFv was constructed by linking the V_(L) (SEQ ID NO:1 and 2)and V_(H) (SEQ ID NO: 3 and 4) domains of human monoclonal antibody B11as shown in FIG. 9. EGF sequences were incorporated in order to detectthe binding of the ScFv to dendritic cells using anti-EFG antibodies.Dendritic cells were incubated with B11-ScFv for one hour at 4° C., thenwashed before incubation with anti-EFG-FITC probe for 1 hour at 4° C.The samples were analyzed using FACS analysis.

The results of the FACS analysis demonstrated that the B11 ScFv fragmentof human monoclonal antibody B11 bound to human dendritic cells.Accordingly, the ScFv can be used as a vaccine or as an immunotoxin bylinking the ScFv to a selected antigen or toxin, respectively.

IX. Binding of F(ab′)2 Fragments of Human Antibody B11 to HumanDendritic Cells

The reactivity of F(ab′)2 fragments of human monoclonal antibody B11with dendritic cells from human tissues was evaluated by flow cytometry.These experiments were also designed to evaluate whether the Fc portionof human antibody B11 is significantly involved in the binding of B11 todendritic cells.

F(ab′)2 fragments were prepared by digestion of purified B11 mAb withpepsin under standard conditions. The F(ab′)2 fragments were purified byprotein L chromatography. Dendritic cells, prepared fresh from humanmonocytes by culture in GM-CSF and IL-4, were incubated with wholeantibody or F(ab′)2 antibody fragments for 1 hour at 4° C. The cellswere washed before incubation with anti-human IgG-F(ab′)2-PE probe for 1hour at 4° C. The cells were washed again prior to analysis using aFACScalibur instrument and Cellquest software.

As shown in FIG. 10, the results of the FACS analysis demonstrated thatwhole human monoclonal antibody B11 and F(ab′)2 fragments of B11 boundto dendritic cells with similar kinetics, indicating that the Fc portionof the whole monoclonal antibody does not contribute significantly tobinding of B11 to dendritic cells.

Example 3 Characterization of B11 Target Antigen

I. Immunoprecipitation of the Human Monoclonal Antibody B11-TargetAntigen from Dendritic Cells

Human monoclonal antibody B11 was used to immunoprecipitate its cognatetarget antigen from dendritic cells.

Briefly, cell lysates from dendritic cells were prepared and incubatedwith monoclonal antibody B11 or an isotype control IgG antibody at 4° C.Antibody-antigen complexes were captured with anti-human IgG-agarose,and separated by SDS polyacrylamide gel electrophoresis.

A band corresponding to a molecular weight of approximately 180kilodaltons was evident in antibody B11 immunoprecipitates from twodifferent preparations of dendritic cell lysates, but not in the controlsample. Accordingly, these immunoprecipitation studies showed that humanmonoclonal antibody B11 recognizes a target antigen on dendritic cellswith an approximate molecular weight of 180 kilodaltons, as analyzed bySDS-PAGE.

II. N-Terminal Sequencing of the Human Monoclonal Antibody B11 TargetAntigen from Dendritic Cells

Following immunoprecipitation as described above, the B11 target antigenwas subjected to N-terminal amino acid sequencing to determine homologywith known proteins.

Cell lysates were prepared from dendritic cells and allowed to incubatewith monoclonal antibody B11 at 4° C. Antibody-antigen complexes werecaptured with anti-human IgG-agarose, and separated by SDSpolyacrylamide gel electrophoresis. Proteins from the gel weretransferred to nitrocellulose and the band corresponding to themonoclonal antibody B11 target antigen was eluted for N-terminal aminoacid sequencing. The N-terminal sequencing and data base search wereconducted at Midwest Analytical, Inc. (St. Louis, Mo.).

Sequencing of the N-terminal 15 amino acid residues of the monoclonalantibody B11 target antigen revealed protein sequence homology with thehuman macrophage mannose receptor, as follows:

B11 antigen (SEQ ID NO: 5) DDXXQFLIXXEDXKR Macrophage mannose receptor(SEQ ID NO: 6) LDTRQFLIYNEDHKR 

A computer search of the human protein database did not identify anyother proteins as having significant homology to B11 antigen.

In a further study, dendritic cell lysates were cleared ofnon-specifically binding proteins by overnight incubation withanti-mouse IgG-charged agarose The agarose was spun out, and the clearedsupernatant was recovered and incubated overnight with anti-humanIgG-agarose previously charged with antibody B11. The agarose was washedwith PBS and boiled with reducing loading buffer. Finally, the agarosewas spun out and the supernatant was loaded onto a gel. Antibody B11immunoprecipitated a protein of approximately 150-180 kD. This proteinwas blotten onto a PVDF membrane and sent to Midwest Analytical, Inc.for microsequencing.

The N-terminal microsequencing results of the monoclonal antibody B11target antigen revealed the following protein sequence: LLDTR QFLIYLEDTK RCVDA (SEQ ID NO:7). This sequence again matched that of the humanmacrophage mannose receptor (GenBank Accession #NP_(—)002429) with 100%identity over 20 amino acids as determined using the BLAST algorithm atthe National Center for Biotechnology Information web site(http://www.ncbi.nlm.nih.gov/). These data indicate that the targetmolecule on dendritic cells recognized by human monoclonal antibody B11is the macrophage mannose receptor.

III. B11 Inhibits FITC-Dextran Uptake by Dendritic Cells

The following experiment was designed to test whether antibody B11blocks the mannose receptor and thus can be used, for example, toprevent or inhibit interaction of pathogens with the mannose receptor(e.g., cellular infection).

Dendritic cells were incubated with FITC-Dextran (500 μg/ml) and eitherisotype control human IgG or B11 HuMAb (25 μg/ml) for 30 minutes at thetemperatures indicated in FIG. 11. Dextran molecules are known to bespecifically internalized by dendritic cells through the mannosereceptor (F. Sallusto et. al. J. Exp. Med. 1995, 185:389-400). Labelled(FITC)-dextran uptake was determined by FACS analysis (i.e.,fluorescence intensity of the dendritic cells samples using aFACScalibur instrument). The percent of FITC-dextran uptake was set at100% at 37° C. and 0% at 4° C.

As shown in FIG. 11, antibody B11 blocked the uptake of FITC-dextran by61.5% under these conditions. These results suggest that humanmonoclonal antibody B11 can be used to block the interaction ofpathogens with the mannose receptor.

Example 4 Activity of Human Anti-Dendritic Cell Antibodies I.Internalization of Human Monoclonal Antibody B11 by Dendritic Cells

The extent to which human antibody B11 is internalized following bindingto dendritic cells was evaluated by flow cytometry.

Dendritic cells were prepared from adherent mononuclear cells asdescribed above. The cells were incubated with a saturatingconcentration of monoclonal antibody B11-FITC at 4° C. for 1 hour toallow maximum surface binding of the antibody, washed with cold PBS toremove excess antibody, and then incubated at 37° C. for various periodsof time to allow for antibody internalization. The samples were thenwashed with cold PBS to stop the reaction, further washed with 0.1% PBA,pH 2.5 to strip surface bound antibody from the cells. The remainingfluorescence is antibody B11-FTIC that has been internalized. Controlcells were immediately washed with 0.1% PBA, pH 2.5 and kept at 4° C. torepresent minimal internalization, or washed only with 0.1% PBA, pH 7 torepresent maximal loading of antibody B11-FITC. The percent antibodyinternalization was calculated by the following formula:

% Internalization=(mean fluorescence intensity of sample−meanfluorescence intensity of acid washed 4° C. control)/(mean fluorescenceintensity of PBS washed 4° C. control−mean fluorescence intensity ofacid washed 4° C. control)

The results, shown in FIG. 7, demonstrate that human monoclonal antibodyB11 is efficiently internalized after binding to dendritic cells. Thisunexpected property of human anti-dendritic cell monoclonal antibody B11indicates that the antibody can be used to deliver agents, such asantigens and toxins, intracellularly to dendritic cells.

II. B11-FITC Internalization by Dendritic Cells

Microscopic visualization was also used to confirm that antibody B11 isinternalized following binding to dendritic cells.

Dendritic cells were generated by incubating monocytes with GM-CSF (10ng/ml) and IL-13 (50 ng/ml) under non-adherent conditions for 7 days.These were combined with mAb B11-FITC (1 μg) in the presence of humanIgG (600 μg) and anti-CD32 mAb IV1.3 (10 μg) to block non-specificuptake and incubated at 37° C. Control cells were incubated on iceduring the entire procedure. After 15 and 60 minutes at 37° C.,dendritic cells were placed on ice and stained with anti-CD11c-PE (1μg). Internalization of mAb B11 was examined by confocal microscopyusing a BioRad MRC1024 laser scanning confocal microscope. Cells werescanned for fluorescence using the 488 nm line from a 15 mW Kr/Ar laserand two photodetectors (522/35 nm dichroic for FITC fluorescence and605/32 nm dichroic for PE fluorescence). A 63× Plan-APO 1.4 NA objective(Carl Zeiss, Inc., Thornwood, N.Y.) was used in conjunction with an irissetting of 2.1 which allowed for detection of optical sections of thefluorescence image that were approximately 1.0 μm thick. Representativeimages were selected from the slices through the center of the cellsafter sectioning the entire cell.

The results confirmed that B11 mAb was rapidly internalized by dendriticcells after binding to the mannose receptor, again suggesting thisantibody can efficiently deliver antigens or toxins into antigenpresenting cells. Consistent with the FACS data, the internalization wasevident within 15 minutes, and nearly complete within one hour.

III. Enhanced Antigen Presentation by Dendritic Cells Following TargetedAntigen Delivery with Human Monoclonal Antibody B11

In order to determine whether human monoclonal antibody B11 can be usedto enhance the processing and presentation of antigens by dendriticcells, the antibody was conjugated to the tetanus toxoid (TT) antigenusing the chemical cross-linking reagent SMCC.

Dendritic cells were prepared from adherent mononuclear cells asdescribed above, with the exception that cells were cultured in Tefloncontainers. Dendritic cells were harvested and replated in 96 wellmicrotiter plates at 5000 cells per well in macrophage serum-free mediumwith 10% fetal calf serum. Monoclonal antibody B11 conjugated to tetanustoxoid or tetanus toxoid alone was added at various concentrations tothe dendritic cells. Autologous tetanus toxoid-specific T cellsgenerated by incubation of mononuclear cells with tetanus toxoidfollowed by IL-2 were added to each well containing dendritic cells at50,000 cells per well. Cells were cultured together for 7 days at 37° C.and assayed for the number of living cells using a MTT based assayaccording to the manufacturer's instructions (Promega, Madison, Wis.).The ability to induce dendritic cells to specifically stimulate tetanustoxoid-specific T lymphocytes was compared after exposing cells totetanus toxoid or antibody B11-tetanus toxoid. The results (FIG. 8)showed that conjugating tetanus toxoid as a model antigen to B11 leadsto significantly more efficient antigen presentation as measured byantigen-specific T cell proliferation.

In another experiment, ³H-Thymadine was used as the readout for T cellproliferation following loading of dendritic cells with either B11conjugated with tetanus toxoid (TT) or with tetanus toxoid mixed withB11 antibody (unconjugated control). As an additional control, ablocking antibody to FcγRII (CD32), mAb IV-3, was added to some wellscontaining the B11-TT conjugate to determine whether this Fc receptorcontributed to enhanced antigen processing and presentation by B11-TT.The following day, cells were combined with freshly thawed, previouslyestablished tetanus toxoid-specific T-cells for 4 days. Cells wereco-incubated with ³H-Thymadine at 37° C. during the final day. Theamount of ³H incorporated into the T cells was assayed.

As with the previous experiment, the results (FIG. 12) demonstrated thatconjugating tetanus toxoid as a model antigen to B11 leads tosignificantly more efficient antigen presentation as measured byantigen-specific T cell proliferation. Addition of excess amounts of mAbIV.3 did not significantly alter the enhanced antigen presentation byB11-TT conjugate, showing that interaction of B11-TT with FcγRII is notrequired for this activity.

Overall, the results of these studies, shown in FIGS. 8 and 12, indicatethat 10 to 100-fold lower amounts of antibody B11-conjugated tetanustoxoid are required to achieve the level of T cell stimulation comparedwith tetanus toxoid alone. In addition, the absolute degree of T cellstimulation as shown in FIG. 8 was 2-fold greater when tetanus toxoidwas targeted to dendritic cells with antibody B11. Thus, datademonstrate that an antigen can be conjugated to human monoclonalantibody B11 and that the antibody targeted antigen is processed andpresented more efficiently than non-targeted antigen, leading toenhanced antigen-specific T cell responses.

Example 5 Characterization of Human Monoclonal Antibody E21 AgainstDendritic Cells I. Molecular Weight Analysis of Human Antibody E21Antigen

Dendritic cell lysates were prepared from cultured human dendriticcells. Briefly, dendritic cells were washed and resuspended in TritonX-100 containing lysis buffer at 4° C. The unfractionated lysate wasloaded onto a 4-15% SDS-polyacrylamide gel and then the protein weretransferred to nitrocellulose. The blot was incubated with 10 ug/ml E21followed by anti-human IgG-alkaline phosphatase probe and visualizedusing horseradish peroxidase.

The results of this experiment demonstrated that the human monoclonalantibody E21 antigen has an approximate molecular weight of 36-40kilodaltons.

II. Binding of Human Antibody E21 to Human Dermal and Epidermal HumanDendritic Cells

As shown in FIG. 1, human monoclonal Ab E21 bound preferentially todendritic cells. This experiment was designed to test the reactivity ofhuman antibody E21 to dermal and epidermal dendritic cells byimmunohistochemistry analysis of frozen skin with E21.

Frozen sections of human skin were stained with FITC-E21 or FITC-huIgGcontrol, and detected using rabbit anti-FITC probe.

The results of the immunohistochemistry analysis demonstrate that humanantibody E21 reacts with both dermal dendritic cells/macrophages andepidermal dendritic cells (Langerhan cells) in human skin sections.

III. Binding of Human Antibody E21 to Macaque Dendritic Cells

The animal (monkey) model of cynomolgus macaques can provide relevantinformation regarding the clinical application of antibodies, providedthat the target antigen is conserved among primates. Accordingly, thecross-reactivity of human monoclonal antibody E21 with dendritic cellsfrom cynomolgus monkey was evaluated by flow cytometry.

Cynomolgus monocytes were differentiated into dendritic cells withGM-CSF and IL-4 treatment, and tested for E21 binding by flow cytometry.The dendritic cells were incubated with E21 for 1 hour at 4° C., thenwashed before incubation with anti-human IgG-FITC probe for 1 hour at 4°C. The samples were analyzed using a FACScalibur instrument.

Conclusion

The foregoing Examples demonstrate the generation of human monoclonalantibodies that specifically react with high affinity to dendriticcells.

In particular, human monoclonal antibody B11 specifically recognizes thehuman macrophage mannose receptor on dendritic cells. In addition, humanmonoclonal antibody B11 is efficiently internalized by dendritic cells,and enhances antigen processing and presentation by dendritic cells.

Human monoclonal antibody E21 binds to a different antigen than B11 onhuman dendritic cells. The E21 antibody also cross-reacts with dendriticcells derived from cynomolgus macaques (monkey), suggesting that the E21antigen is conserved in primates and therefore provides a relevantanimal model for further development of the antibody.

These results support the conclusion that the fully human monoclonalantibodies of the present invention, including fragments, conjugates andbispecific molecules thereof, can be used are for the diagnosis andtreatment of dendritic cell related disorders.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-23. (canceled)
 24. A method of targeting an antigen to a dendriticcell comprising contacting the cell with an antibody, or antigen-bindingportion thereof, that binds to the human macrophage mannose receptor,wherein the antibody, or antigen-binding portion thereof, (a) is linkedto an antigen and (b) comprises heavy and light chain variable regionsas shown in SEQ ID NO:2 and SEQ ID NO:4, respectively.
 25. A method ofinducing or enhancing an immune response against an antigen in a subjectcomprising administering to the subject an antibody, or anantigen-binding portion thereof, that binds to the human macrophagemannose receptor, wherein the antibody, or an antigen-binding portionthereof, (a) is linked to an antigen and (b) comprises heavy and lightchain variable regions as shown in SEQ ID NO:2 and SEQ ID NO:4,respectively.
 26. A method of immunizing a subject comprisingadministering to the subject an antibody, or antigen-binding portionthereof, that binds to the human macrophage mannose receptor, whereinthe antibody, or antigen-binding portion thereof, (a) is linked to anantigen and (b) comprises heavy and light chain variable regions asshown in SEQ ID NO:2 and SEQ ID NO:4, respectively.
 27. The method ofclaim 25, wherein the immune response comprises presentation of theantigen as a component of an MHC-I or MHC-II conjugate.
 28. The methodof claim 26, wherein the antibody is administered in an amountsufficient to induce cytokine release by dendritic cells.
 29. The methodof any one of claims 24-26, wherein the antigen is a tumor antigen, anautoantigen, or a component of a pathogen.
 30. The method of any one ofclaims 24-26, wherein the antigen is selected from the group of antigensconsisting of Pmel-17, HER2/neu, CEA, gp100, MART1, TRP-2, melan-A,NY-ESO-1, MN (gp250), idiotype, MAGE-1, MAGE-3, Tyrosinase, Telomerase,and MUC-1.
 31. The method of any one of claims 24-26, wherein theantibody, or an antigen-binding portion thereof, is linked to theantigen to form a molecular conjugate which is produced as a recombinantfusion protein or a chemical conjugate or is encoded by the nucleotidesequence shown in SEQ ID NO:8.